title: Internalization studies of chimeric neuropeptide Y receptors Y1 and Y2 suggest complex interactions between cytoplasmic domains.
abstract: Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory results for the Y2 receptor. In order to explore putative functional differences between the Y1 and Y2 receptors we generated reciprocal chimeras by swapping the third intracellular loop, the carboxy terminus or both between human Y1 and Y2. Internalization was studied in a quantitative radioligand binding assay with removal of surface-bound ligand in an acidic-wash procedure. The internalization assay revealed a lower degree of internalization as well as slower kinetics for the Y2 receptor. Generally, reciprocal exchange of receptor segments did not convey properties of the donor receptor but tended to enhance internalization. Surprisingly, insertion of the Y2 carboxy terminus into Y1 gave almost complete internalization (92%), rather than reduced internalization, while the insertion of both segments resulted in internalization equal to the native Y1 receptor. These findings were confirmed by fluorescence microscopy of immuno-stained receptors tagged with a C-terminal FLAG epitope. However, after exposure to high agonist concentrations (100 nM) Y2 was internalized. Studies of Y2 and the closely related Y7 receptor confirmed low internalization for Y2 from chicken and teleost fishes as well as Y7 from two teleosts. The conservation across species of low internalization at physiological concentrations suggests that this is an ancient feature and of vital importance for Y2 function. We propose that amino acid motifs in the third intracellular loop as well as the C terminus of both Y1 and Y2 are able to drive agonist-promoted internalization and that there may be constraining motifs in the Y2 receptor.
authors.affiliation: Department of Neuroscience, Uppsala University, Sweden. Ingrid.Lundell@neuro.uu.se
authors.forename: Ingrid
authors.initials: I
authors.lastname: Lundell
authors.forename: Nadine
authors.initials: N
authors.lastname: Rabe Bernhardt
authors.forename: Anna-Karin
authors.initials: AK
authors.lastname: Johnsson
authors.forename: Dan
authors.initials: D
authors.lastname: Larhammar
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D003593:Cytoplasm / Q000378:metabolism*; D005399:Fishes; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017476:Receptors, Neuropeptide Y / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; C097212:neuropeptide Y-Y1 receptor; C095889:neuropeptide Y2 receptor