Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y(1) receptors.
The human NPY Y(1) 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, Y(1) receptors lacking the last 32 C-terminal amino acids (Y(1)Δ32) are constitutively internalized, unlike full-length Y(1) receptors. At steady state, internalized Y(1)Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y(1)Δ32 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 (Y(1)Δ42) 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 Y(1)Δ32 receptors. We show that the agonist-independent internalization of Y(1)Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y(1) receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y(1)Δ32 receptors. We suggest that this motif is masked in full-length Y(1) receptors which do not constitutively internalize in the absence of agonist.
Lecat
Sandra
S
Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, FRE 3211, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, Illkirch, France.
Ouédraogo
Moussa
M
Cherrier
Thomas
T
Noulet
Fanny
F
Rondé
Philippe
P
Glasser
Nicole
N
Galzi
Jean-Luc
JL
Mely
Yves
Y
Takeda
Kenneth
K
Bucher
Bernard
B
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D017476:Receptors, Neuropeptide Y / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014168:Transferrin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D002966:Clathrin; D009478:Neuropeptide Y; D034741:RNA, Small Interfering; D017476:Receptors, Neuropeptide Y; D014168:Transferrin; C097212:neuropeptide Y-Y1 receptor; D014443:Tyrosine; D020696:rab5 GTP-Binding Proteins