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.
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004705:Endocytosis; D017930:Genes, Reporter; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009154:Mutation; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry; D021381:Protein Transport; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000172:deficiency / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection
D008024:Ligands; D010446:Peptide Fragments; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor