The RAMP interactome reveals a key role for MYO6 in regulating CLR-RAMP signaling

A therapeutically tractable class of proteins implicated in lymphangiogenesis, the growth of lymphatic cells, are G-protein coupled receptors (GPCRs) and their associated receptor-activity modifying proteins (RAMPs). RAMPs regulate GPCR trafficking and signaling. Adrenomedullin (ADM), a pro-lymphangiogenic ligand associated with lymphedema, and calcitonin gene-related peptide (CGRP), a peptide implicated with migraine pathology, are both ligands that bind to calcitonin receptor-like receptor (CLR) and lead to receptor internalization. The ADM receptor consists of the heterodimerization of CLR with either RAMP2 or 3 while the CGRP receptor consists of the heterodimerization of CLR with RAMP1. MYO6 is a molecular motor protein involved in endocytosis. BIO ID-based proteomics discovered that MYO6 was enriched in the presence of CLR and RAMP2. Upon knockdown of MYO6 in Lymphatic Endothelial Cells (LECs), treatment with ADM or CGRP resulted in impaired CLR-RAMP2/RAMP3 ADM and CLR-RAMP1 CGRP signaling. However, it remains unclear if the signaling phenotype is due to 1) fewer receptor complexes available to signal at the plasma membrane or (2) impaired endosomal signaling. Our hypothesis is that loss of MYO6 impairs CLR-RAMP internalization, thereby inhibiting endosomal signaling to result in decreased intracellular signaling pathways (pAKT and pERK). To test this hypothesis, we performed a BRET1-based β-arrestin recruitment assay, which is upstream of receptor endocytosis. We found that loss of MYO6 did not impact β- arrestin recruitment to agonist stimulated CLR-RAMP complexes, suggesting that receptor availability remained unaffected by the loss of MYO6. Paradoxically, when we blocked endocytosis using an inhibitor of dynamin1/2 (Dynasore), we saw increased CLR signaling, which did not mimic the effects of knockdown of MYO6. This is likely due to prolonged β- arrestin-independent signaling from an uncleaved clathrin-coated pit. By understanding the role MYO6 plays as well as normal CLR functioning and internalization, it is possible to develop treatments and therapies for diseases underlying the lymphatic system and that affect lymphangiogenesis.