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ORIGINAL ARTICLE

Role of the C Terminus in Neuropeptide Y Y Receptor Desensitization and Internalization

Wolfson Centre for Age-Related Diseases, King's College London, Guy's Campus, London Bridge, London, United Kingdom

Abstract

We have studied truncation mutants of the rat neuropeptide Y (NPY) Y₁ receptor lacking four (Thr361stop, Y1T361*) or eight (Ser352stop, Y1S352*) potential serine/threonine C-terminal phosphorylation sites. NPY-stimulated hemagglutinin-tagged Y1, Y1T361*, and Y1S352* receptors all efficiently activated G proteins in Chinese hamster ovary (CHO) cell membranes, but desensitization after NPY pretreatment 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, whereas 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. It is surprising that both truncated receptors were constitutively internalized, predominantly in transferrin-positive compartments. NPY increased cell-surface localization of HAY1S352* receptors, whereas the distribution of both mutants was unaltered by BIBO3304. Recruitment of green fluorescent protein-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.

Address correspondence to: Dr. Nicholas Holliday, Neuronal Receptors and Signaling Group, Wolfson CARD, King's College London, Guy's Campus, 19 Newcomen Street, London Bridge, London SE1 1UL, UK. E-mail: nicholas.2.holliday{at}kcl.ac.uk

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