Ligand-induced internalization, recycling, and resensitization of adrenomedullin receptors depend not on CLR or RAMP alone but on the receptor complex as a whole

• All combinations of AM receptors (CLR–RAMP) were quickly internalized after ligand induction.
• Recycling rates of the internalized CLR–RAMP complexes depend on both partners.
• AM receptor complexes recycled to the plasma membrane were functionally active.
• The efficacies of recycled receptors are different for each AM receptor complex.
• The ligand-induced events of AM receptors are dictated not only by RAMPs but also by core GPCR.

Adrenomedullins (AM) is a multifaceted distinct subfamily of peptides that belongs to the calcitonin gene-related peptide (CGRP) superfamily. These peptides exert their functional activities via associations of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs) RAMP2 and RAMP3. Recent studies established that RAMPs and CLRs can modify biochemical properties such as trafficking and glycosylation of each other. However there is very little or no understanding regarding how RAMP or CLR influence ligand-induced events of AM-receptor complex. In this study, using pufferfish homologs of CLR (mfCLR1–3) and RAMP (mfRAMP2 and mfRAMP3), we revealed that all combinations of CLR and RAMP quickly underwent ligand-induced internalization; however, their recycling rates were different as follows: mfCLR1–mfRAMP3 > mfCLR2–mfRAMP3 > mfCLR3–mfRAMP3. Functional receptor assay confirmed that the recycled receptors were resensitized on the plasma membrane. In contrast, a negligible amount of mfCLR1–mfRAMP2 was recycled and reconstituted. Immunocytochemistry results indicated that the lower recovery rate of mfCLR3–mfRAMP3 and mfCLR1–mfRAMP2 was correlated with higher proportion of lysosomal localization of these receptor complexes compared to the other combinations. Collectively our results indicate, for the first time, that the ligand-induced internalization, recycling, and reconstitution properties of RAMP–CLR receptor complexes depend on the receptor-complex as a whole, and not on individual CLR or RAMP alone.