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)Delta32) are constitutively internalized, unlike full-length Y(1) receptors. At steady state, internalized Y(1)Delta32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y(1)Delta32 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)Delta42) 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)Delta32 receptors. We show that the agonist-independent internalization of Y(1)Delta32 receptors involves a tyrosine-based motif YXXPhi. 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)Delta32 receptors. We suggest that this motif is masked in full-length Y(1) receptors which do not constitutively internalize in the absence of agonist.