Implication of various forms of neurotensin receptors in the mechanism of internalization of neurotensin in cerebral neurons.
This report describes the kinetics and molecular aspects of neurotensin internalization in neurons. Incubation of alpha-125I-Bolton-Hunter neurotensin-(2-13) with cortical neurons at 37 degrees C was followed by a rapid internalization of the peptide bound to its receptors. This process was completed within 20 min and was inhibited either irreversibly by the general endocytosis inhibitor phenylarsine oxide or reversibly by incubation at low temperature (0-4 degrees C). The discrepancy of maximal binding capacities observed in the presence (150 fmol/mg of protein) or in the absence (250 fmol/mg of protein) of internalization inhibitors could be attributed to the appearance of a new pool of neurotensin binding sites on the cell surface rather than a recycling of internalized receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in denaturing conditions revealed that three different protein subunits of 50, 60, and 100 kDa were covalently labeled at 37 degrees C with a radioactive photoreactive analogue of neurotensin. The 50- and 60-kDa subunits remained labeled when internalization was blocked, whereas the specific labeling of the 100-kDa protein was abolished. These results suggest that neurotensin-induced internalization of the 50- and 60-kDa subunits initially present on the cell surface triggers insertion of the 100-kDa subunit into the membrane from an intracellular compartment. Subcellular fractionation experiments have shown that, in the absence of neurotensin, the 100-kDa protein is located in an intracellular vesicular fraction of neurons.
Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice-Sophia Antipolis, Valbonne, France.
J
J
Chabry
G
G
Gaudriault
J P
JP
Vincent
J
J
Mazella
D000818:Animals; D001667:Binding, Competitive; D002454:Cell Differentiation; D002540:Cerebral Cortex / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D009496:Neurotensin / Q000378:metabolism*; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D009496:Neurotensin