Role of the C terminus in neuropeptide Y Y1 receptor desensitization and internalization.
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 beta-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 beta-arrestin proteins. However, additional beta-arrestin-independent mechanisms control Y1 receptor trafficking under basal conditions.
Holliday
Nicholas D
ND
Wolfson Centre for Age-Relatated Diseases, King's College London, Guy's Campus, 19 Newcomen Street, London Bridge, London SE1 1UL, UK. nicholas.2.holliday@kcl.ac.uk
Lam
Chi-Wing
CW
Tough
Iain R
IR
Cox
Helen M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism; D017476:Receptors, Neuropeptide Y / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D009478:Neuropeptide Y; D010446:Peptide Fragments; D011964:Receptors, Gastrointestinal Hormone; D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor; C082172:peptide YY receptor; D016244:Guanosine 5'-O-(3-Thiotriphosphate)