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Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y1 receptors
Authors:Sandra Lecat,Moussa Oué  draogo,Thomas CherrierFanny Noulet,Philippe Rondé  Nicole Glasser,Jean-Luc GalziYves Mely,Kenneth TakedaBernard Bucher
Affiliation:
  • a UMR 7213, CNRS/Université de Strasbourg, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, 74 route du Rhin, BP 60024, 67401 Illkirch, France
  • b Institut de Recherche de l''Ecole de Biotechnologie de Strasbourg, FRE 3211, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67412 Illkirch, France
  • 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.
    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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