Thr-422 and Tyr-424 residues in the carboxyl terminus are critical for the internalization of the rat neurotensin receptor.
In order to identify the amino acid sequences responsible for the internalization of the cloned rat brain neurotensin receptor, we carried out site-directed mutagenesis of the cDNA encoding the receptor followed by expression of the receptor into mammalian COS 7 cells. In cells transfected with the full-length neurotensin receptor, 56% of iodinated neurotensin specifically bound to the cells after 60 min of incubation at 37 degrees C was internalized. Deletions made in the third intracellular loop did not affect receptor internalization. By contrast, internalization was reduced to 5% of total in cells in which almost all the carboxyl-terminal tail of the receptor had been deleted (R392stop). In order to determine which part of the tail was responsible for this effect, several Ser and Thr residues were deleted in the carboxyl cytoplasmic sequence of the receptor. Almost all of these receptors were internalized as efficiently as the wild type. Only the form of the neurotensin receptor truncated at Glu-421 (deletion of the last three residues, TLY) produced a significant decrease in the amount of ligand internalized. Finally, point mutations of Thr-422 and Tyr-424 residues to Gly led to an almost complete loss of ligand internalization demonstrating the involvement of these 2 residues in the internalization process. Replacement of the last three amino acids by the cytoplasmic endocytosis signal of the vesicular stomatitis virus did not restore the efficiency of neurotensin receptor internalization. These biochemical results were confirmed by confocal microscopic analysis. Cell transfected with the wild type receptor showed a temperature-dependent intracellular accumulation of a fluorescent analog of neurotensin, whereas cells transfected with a receptor truncated at the carboxyl terminus showed a clustering of the fluorescent peptide at the cell surface.
Chabry
J
J
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, UPR 411, Sophia Antipolis, Valbonne, France.
Botto
J M
JM
Nouel
D
D
Beaudet
A
A
Vincent
J P
JP
Mazella
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D018028:Receptors, Neurotensin / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D018028:Receptors, Neurotensin; D013912:Threonine; D014443:Tyrosine; D003545:Cysteine