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 mechanism s underlying this process, we monitored in parallel the migration of the NT 1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalizatio n was prevented by hgpertonic sucrose, potassium depletion and cytosol acid ification, demonstrating that it proceeded via clathrin-coated pits, Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine O range-positive, acidic organelles, These organelles concentrated transferri n and immunostained positively for rab 5A, therefore they were early endoso mes, 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 n onacidic juxtanuclear compartment identified as the Trans-Golgi Network (TG N) 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 recruite d to the TGN from either late or recycling endosomes, By that time, interna lized receptors were detected in Lamp-l-immunoreactive lysosomes, These res ults 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 prote in from endosomes to the TGN.