title: Ligand-induced internalization, recycling, and resensitization of adrenomedullin receptors depend not on CLR or RAMP alone but on the receptor complex as a whole.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan; Department of Pathology and Molecular Medicine, McMaster University, 1200 Main St. W. Hamilton, ON L8N 3Z5, Canada; Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Electronic address: kakonpoly@yahoo.com.
authors.forename: Kakon
authors.initials: K
authors.lastname: Nag
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Naznin
authors.initials: N
authors.lastname: Sultana
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kato
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, 1200 Main St. W. Hamilton, ON L8N 3Z5, Canada.
authors.forename: Anna
authors.initials: A
authors.lastname: Dranik
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Nakamura
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kutsuzawa
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Shigehisa
authors.initials: S
authors.lastname: Hirose
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Akaike
mesh_terms: D053607:Adrenomedullin / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D015740:Calcitonin Gene-Related Peptide; D058286:Calcitonin Receptor-Like Protein / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005399:Fishes; D005434:Flow Cytometry; D006031:Glycosylation; D007124:Immunoenzyme Techniques; D008024:Ligands; D010446:Peptide Fragments / Q000378:metabolism*; D021381:Protein Transport; D058263:Receptor Activity-Modifying Protein 2 / Q000378:metabolism*; D058264:Receptor Activity-Modifying Protein 3 / Q000378:metabolism*; D058265:Receptors, Adrenomedullin / Q000378:metabolism*
chemical_list: D058286:Calcitonin Receptor-Like Protein; D008024:Ligands; D010446:Peptide Fragments; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058265:Receptors, Adrenomedullin; D053607:Adrenomedullin; D015740:Calcitonin Gene-Related Peptide
keywords: Adrenomedullin; CGRP; CLR; GPCR; RAMP; Signaling