title: Fluorescently labeled adrenomedullin allows real-time monitoring of adrenomedullin receptor trafficking in living cells.
abstract: 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 (AM1), 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/tBu 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 AM1 internalization behavior.
authors.affiliation: Institut für Biochemie, Universität Leipzig, Brüderstraße 34, 04103, Leipzig, Germany.
authors.forename: Ria
authors.initials: R
authors.lastname: Schönauer
authors.affiliation: Institut für Biochemie, Universität Leipzig, Brüderstraße 34, 04103, Leipzig, Germany.
authors.forename: Anette
authors.initials: A
authors.lastname: Kaiser
authors.affiliation: Institut für Biochemie, Universität Leipzig, Brüderstraße 34, 04103, Leipzig, Germany.
authors.forename: Cathleen
authors.initials: C
authors.lastname: Holze
authors.affiliation: Institut für Biochemie, Universität Leipzig, Brüderstraße 34, 04103, Leipzig, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Babilon
authors.affiliation: Bayer Pharma AG, Aprather Weg 18A, 42113, Wuppertal, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Köbberling
authors.affiliation: Bayer Pharma AG, Aprather Weg 18A, 42113, Wuppertal, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Riedl
authors.affiliation: Institut für Biochemie, Universität Leipzig, Brüderstraße 34, 04103, Leipzig, Germany.
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
mesh_terms: D053607:Adrenomedullin / Q000737:chemistry* / Q000378:metabolism; D058286:Calcitonin Receptor-Like Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000648:ultrastructure; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D058263:Receptor Activity-Modifying Protein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058265:Receptors, Adrenomedullin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry
chemical_list: C507862:ADM protein, human; C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D005456:Fluorescent Dyes; D010455:Peptides; C545503:RAMP2 protein, human; D058263:Receptor Activity-Modifying Protein 2; D058265:Receptors, Adrenomedullin; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D053607:Adrenomedullin; C005358:tetramethylrhodamine
keywords: ADM receptor; G protein-coupled receptor (GPCR) trafficking; adrenomedullin (ADM); copper(I)-catalyzed azide alkyne cycloaddition (CuAAC); fluorescently labeled peptide; live cell fluorescence microscopy; receptor activity-modifying protein 2 (RAMP2); solid-phase peptide synthesis (SPPS)