Most G-protein-coupled receptors that undergo agonist-dependent internalization require the presence of specific cytoplasmic-tail residues to initiate interactions with proteins of the endocytic machinery. Here we show that the UT receptor (urotensin II receptor) undergoes internalization, and that specific serine residues of the receptor's cytoplasmic tail participate in this process. We first observed a time-dependent increase in internalization of the UT receptor expressed in COS-7 cells following binding of the agonist urotensin II. This sequestration was significantly reduced in the presence of sucrose, demonstrating that the agonist-activated UT receptor is internalized in part by clathrin-coated pits. Moreover, the sequestered receptor was co-localized in endocytic vesicles with β-arrestin1 and β-arrestin2. To assess whether specific regions of the receptor's cytoplasmic tail were involved in internalization, five UT receptor mutants were constructed. In four constructs the receptor's cytoplasmic tail was truncated at various positions (UTΔ367, UTΔ363, UTΔ350 and UTΔ336), and in the other four adjacent serine residues at positions 364–367 were replaced by Ala (Mut4S). Each mutant, except UTΔ367, demonstrated a significantly reduced internalization rate, thereby revealing the importance of specific serine residues within the cytoplasmic tail of the UT receptor for its ability to be internalized efficiently.
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Research Article|
December 14 2004
Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization
Christophe D. PROULX;
Christophe D. PROULX
*Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
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May SIMAAN;
May SIMAAN
†Hormones and Cancer Research Unit, McGill University Health Centre, 687 Pine Avenue West, Montreal, Canada H3A 1A1
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Emanuel ESCHER;
Emanuel ESCHER
*Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
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Stéphane A. LAPORTE;
Stéphane A. LAPORTE
†Hormones and Cancer Research Unit, McGill University Health Centre, 687 Pine Avenue West, Montreal, Canada H3A 1A1
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Gaétan GUILLEMETTE;
Gaétan GUILLEMETTE
*Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
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Richard LEDUC
Richard LEDUC
1
*Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
1To whom correspondence should be addressed (email Richard.Leduc@USherbrooke.ca).
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Publisher: Portland Press Ltd
Received:
May 13 2004
Revision Received:
September 28 2004
Accepted:
September 30 2004
Accepted Manuscript online:
September 30 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (1): 115–123.
Article history
Received:
May 13 2004
Revision Received:
September 28 2004
Accepted:
September 30 2004
Accepted Manuscript online:
September 30 2004
Citation
Christophe D. PROULX, May SIMAAN, Emanuel ESCHER, Stéphane A. LAPORTE, Gaétan GUILLEMETTE, Richard LEDUC; Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization. Biochem J 1 January 2005; 385 (1): 115–123. doi: https://doi.org/10.1042/BJ20040807
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