Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics
We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position -4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.
|3T3 Cells, Animals, Binding Sites, COS Cells, Cercopithecus aethiops, Endocytosis, Genes, Reporter, Granulocyte Colony-Stimulating Factor/*pharmacology, Hela Cells, Humans, Kinetics, Ligands, Mice, Mice, Knockout, Microscopy, Confocal, Mutation, Organelles/metabolism/ultrastructure, Peptide Fragments/chemistry, Protein Transport, Receptors, Granulocyte Colony-Stimulating Factor/*chemistry/deficiency/*metabolism, Recombinant Fusion Proteins/chemistry/metabolism, Research Support, Non-U.S. Gov't, Restriction Mapping, Sequence Deletion, Transfection|
|Organisation||Erasmus MC: University Medical Center Rotterdam|
Aarts, L.H.J, Roovers, O, Ward, A.C, & Touw, I.P. (2004). Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics. Blood. Retrieved from http://hdl.handle.net/1765/8248