Recycling ability of the mouse and the human neurotensin type 2 receptors depends on a single tyrosine residue.
Receptor recycling plays a key role in the modulation of cellular responses to extracellular signals. The purpose of this work was to identify residues in G-protein coupled neurotensin receptors that are directly involved in recycling. Both the high affinity receptor-1 (NTR1) and the levocabastine-sensitive NTR2 are internalized after neurotensin binding. Here, we show that only the mouse NTR2 recycled to the plasma membrane, whereas the rat NTR1 and the human NTR2 did not. Using site-directed mutagenesis, we demonstrate that tyrosine 237 in the third intracellular loop is crucial for recycling of the mouse NTR2. We show that the mouse NTR2 is phosphorylated on tyrosine residues by NT. This phosphorylation is essential for receptor recycling since the tyrosine kinase inhibitor genistein blocks this process. The absence of recycling observed with the human NTR2 could be completely explained by the presence of a cysteine instead of a tyrosine in position 237. Indeed, substitution of this cysteine by a tyrosine gave a mutant receptor that has acquired the ability to recycle to the cell surface after neurotensin-induced internalization. This work demonstrates that a single tyrosine residue in the third intracellular loop of a G-protein-coupled receptor is responsible for receptor phosphorylation and represents an essential structural element for receptor recycling.
Martin
Stéphane
S
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Unité Mixte de Recherche 6097, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France.
Vincent
Jean-Pierre
JP
Mazella
Jean
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D009496:Neurotensin / Q000378:metabolism; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D018028:Receptors, Neurotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; C490985:NTSR2 protein, human; C490984:Ntsr2 protein, mouse; D011500:Protein Synthesis Inhibitors; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D009496:Neurotensin; D014443:Tyrosine; D008985:Monensin; D019833:Genistein