Premium
Fluorescently labeled adrenomedullin allows real‐time monitoring of adrenomedullin receptor trafficking in living cells
Author(s) -
Schönauer Ria,
Kaiser Anette,
Holze Cathleen,
Babilon Stefanie,
Köbberling Johannes,
Riedl Bernd,
BeckSickinger Annette G.
Publication year - 2015
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.2833
Subject(s) - internalization , chemistry , adrenomedullin , peptide , receptor , calcitonin receptor , calcitonin , peptide synthesis , ligand (biochemistry) , g protein coupled receptor , microbiology and biotechnology , biochemistry , calcitonin gene related peptide , biology , neuropeptide , endocrinology
The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM 1 ), a heterodimer consisting of the calcitonin receptor‐like receptor (CLR), a G protein‐coupled receptor, associated with the receptor activity‐modifying protein 2 (RAMP2). Full length and N‐terminally shortened ADM peptides were synthesized using Fmoc/ t Bu solid phase peptide synthesis and site‐specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)‐catalyzed azide alkyne cycloaddition. For the first time, Tam‐labeled ligands allowed the observation of co‐internalization of the whole ligand‐receptor complex in living cells co‐transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2‐complex is routed to the degradative pathway. Moreover, we found that the N‐terminus of ADM is not a crucial component of the peptide sequence in terms of AM 1 internalization behavior. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.