Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.
The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.
Montreal Neurological Institute, McGill University, Montreal, Quebec, H2A 2B4 Canada.
F
F
Vandenbulcke
D
D
Nouel
J P
JP
Vincent
J
J
Mazella
A
A
Beaudet
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D019556:COS Cells; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions / Q000494:pharmacology; D008024:Ligands; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D008954:Models, Biological; D009496:Neurotensin / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D050765:Qa-SNARE Proteins; D018028:Receptors, Neurotensin / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000032:analysis
D015703:Antigens, CD; D002966:Clathrin; D006982:Hypertonic Solutions; D008024:Ligands; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D018028:Receptors, Neurotensin; D014168:Transferrin; D009496:Neurotensin; D013395:Sucrose; D020691:rab GTP-Binding Proteins; D011188:Potassium