TY - JOUR
T1 - Engineered hyperphosphorylation of the b2- Adrenoceptor prolongs arrestin-3 binding and induces arrestin internalization
AU - Zindel, Diana
AU - Butcher, Adrian J.
AU - Al-Sabah, Suleiman
AU - Lanzerstorfer, Peter
AU - Weghuber, Julian
AU - Tobin, Andrew B.
AU - Bünemann, Moritz
AU - Krasel, Cornelius
N1 - Funding Information:
This work was supported by the Deutsche Forschungsgemeinschaft as part of the project A13 of the SFB593 "Mechanisms of cellular compartimentalisa-tion and the relevance for disease." Part of this work was supported by a BBSRC new investigator grant to C.K. [Grant BB/D012902/1]. D.Z. was the recipient of a DAAD PROMOS Travel Grant. A.J.B. and A.B.T. are funded by a MRC program leader grant through the MRC Toxicology Unit. dx.doi.org/10.1124/mol.114.095422. s This article has supplemental material available at molpharm.aspetjournals. org.
Publisher Copyright:
© 2015 by The American Society for Pharmacology and Experimental Therapeutics.
Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 2015/2
Y1 - 2015/2
N2 - G protein-coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C- Terminal tail of the β2- Adrenoceptor (β2AR) and demonstrate that this mutant, termed β2ARSSS, showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the β2AR, whereas the interaction with β2ARSSS was 2- To 4-fold prolonged. In contrast, arrestin-3 interaction with a β2- Adrenoceptor fused to the carboxyl- Terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the b2- Adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild- Type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, β2ARSSS internalized more efficiently than the wild- Type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor- Arrestin affinity are sufficient to alter arrestin trafficking.
AB - G protein-coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C- Terminal tail of the β2- Adrenoceptor (β2AR) and demonstrate that this mutant, termed β2ARSSS, showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the β2AR, whereas the interaction with β2ARSSS was 2- To 4-fold prolonged. In contrast, arrestin-3 interaction with a β2- Adrenoceptor fused to the carboxyl- Terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the b2- Adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild- Type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, β2ARSSS internalized more efficiently than the wild- Type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor- Arrestin affinity are sufficient to alter arrestin trafficking.
KW - Amino Acid Sequence
KW - Arrestins/genetics
KW - Endocytosis/physiology
KW - HEK293 Cells
KW - Humans
KW - Molecular Sequence Data
KW - Phosphorylation/physiology
KW - Protein Binding/physiology
KW - Protein Engineering/methods
KW - Protein Transport/physiology
KW - Receptors, Adrenergic, beta-2/genetics
UR - http://www.scopus.com/inward/record.url?scp=84979860439&partnerID=8YFLogxK
U2 - 10.1124/mol.114.095422
DO - 10.1124/mol.114.095422
M3 - Article
C2 - 25425623
SN - 0026-895X
VL - 87
SP - 349
EP - 362
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 2
ER -