Ligand internalization by cloned neuropeptide Y Y5 receptors excludes Y2 and Y4 receptor-selective peptides

Abstract

In human embryonic kidney-293 (HEK-293) cells, the cloned human neuropeptide Y Y5 receptor saturably internalized agonists, with the rank order of neuropeptide Y-(19-23)-[Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34]human pancreatic polypeptide (neuropeptide Y-Aib-pancreatic polypeptide)>human neuropeptide Y>porcine peptide YY>[Pro34]human peptide YY>[Leu31,Pro34]human peptide YYmuch greater-thanhuman peptide YY-(3-36). Human pancreatic polypeptide competed [125I]neuropeptide Y binding and internalization in neuropeptide Y Y5 receptor-expressing cells, but itself showed no internalization. The internalization was strongly dependent on temperature. The surface binding, and especially the internalization, of human neuropeptide Y were highly sensitive to the clathrin network inhibitor phenylarsine oxide, and to the cholesterol-complexing antibiotic filipin III. The internalized ligands were present in particles corresponding to secondary endosomes in Percoll gradients, but especially in particles banding with the acid hexosaminidase lysosomal marker. At any temperature tested, internalization of the neuropeptide Y Y5 receptor driven by human neuropeptide Y in HEK-293 cells was much slower than the internalization of the neuropeptide Y Y1 receptor reported in the same cells, or in Chinese hamster ovary (CHO) cells. The neuropeptide Y Y5receptor subtype could be the metabotropic receptor responding to protracted challenges by neuropeptide Y-like peptides, and its density could be little sensitive to concentration of extracellular agonists.