Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y1 receptors |
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Authors: | Sandra Lecat,Moussa Oué draogo,Thomas CherrierFanny Noulet,Philippe Rondé Nicole Glasser,Jean-Luc GalziYves Mely,Kenneth TakedaBernard Bucher |
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Affiliation: | a UMR 7213, CNRS/Université de Strasbourg, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, 74 route du Rhin, BP 60024, 67401 Illkirch, Franceb Institut de Recherche de l''Ecole de Biotechnologie de Strasbourg, FRE 3211, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67412 Illkirch, France |
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Abstract: | The human NPY Y1 receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y1 receptors lacking the last 32 C-terminal amino acids (Y1Δ32) are constitutively internalized, unlike full-length Y1 receptors. At steady state, internalized Y1Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y1Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y1Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y1Δ32 receptors. We show that the agonist-independent internalization of Y1Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y1 receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y1Δ32 receptors. We suggest that this motif is masked in full-length Y1 receptors which do not constitutively internalize in the absence of agonist. |
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Keywords: | NPY, neuropeptide Y EGFP, enhanced green fluorescent protein GPCR, G protein-coupled receptor Gi/o, inhibitory GTP binding protein of adenylyl cyclase HEK, human embryonic kidney SP, signal peptide LIC, ligation independent cloning PBS, phosphate buffered saline BSA, bovine serum albumin |
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