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Received for publication August 16, 2004.
Revised November 10, 2004.
Accepted for publication November 30, 2004.
We have studied truncation mutants of the rat neuropeptide Y (NPY) Y1 receptor lacking four (Thr361stop, Y1T361*), or eight (Ser352stop, Y1S352*) potential Ser / Thr C terminal phosphorylation sites. NPY-stimulated haemagglutinin (HA)-tagged Y1, Y1T361* and Y1S352* receptors all efficiently activated G proteins in Chinese hamster ovary (CHO) cell membranes, but desensitization following NPY pre-treatment was only prevented in the HAY1S352* clone. In transfected colonic carcinoma epithelial layers, functional Y1 and Y1T361* peptide YY responses became more transient as the agonist concentration increased, while those mediated by the Y1S352* receptor remained sustained. NPY-stimulated HAY1 receptor phosphorylation was increased by transient overexpression of G protein coupled receptor kinase 2, and only Ser352stop truncation abolished this response in CHO or human embryonic kidney (HEK) 293 cells. Rapid internalization of cell surface HAY1 receptors in HEK293 cells was observed in response to agonist, resulting in partial colocalization with transferrin, a marker for clathrin-mediated endocytosis and recycling. Surprisingly both truncated receptors were constitutively internalized, predominantly in transferrin-positive compartments. NPY increased cell surface localization of HAY1S352* receptors, while the distribution of both mutants was unaltered by BIBO3304. Recruitment of green fluorescent protein (GFP)-tagged 
-arrestin2 to punctate endosomes was observed only for HAY1 and HAY1T361* receptors, and solely under NPY-stimulated conditions. Thus the key C terminal sequence between Ser352 and Lys360 is a major site for Y1 receptor phosphorylation, is critical for its desensitization and contributes to the association between the receptor and -arrestin proteins. However, additional -arrestin independent mechanisms control Y1 receptor trafficking under basal conditions.
Key words:
NPY, Gi family, Desensitization/uncoupling, Sequestration/Internalization, GRKs, barrestins, Neuropeptides, peptidases
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