Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y1 receptors

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Résumé

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.

Mots-clés

Neuropeptide Y Y1 receptor G protein-coupled receptors Internalization Constitutive internalization Recycling