期刊
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
卷 1859, 期 5, 页码 698-711出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2017.01.008
关键词
GPCR dimerization; GPCR traffic; Bimolecular fluorescence complementation assay (BiFC); Split EGFP; Ste2p
资金
- NIH [GM112496]
- International Reintegration Grants Marie Curie Actions [PIRG07-GA-2010-268336]
- Tubitak [110T414, 2214-A]
Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus. Bimolecular Fluorescence Complementation (BiFC) assay was used to determine where receptor dimerization occurred during protein trafficking by monitoring generation of EGFP fluorescence, which occurred upon GPCR dimerization. Our results suggest that these tagged receptors traffic to the membrane as monomers, undergo dimerization or higher ordered oligomerization predominantly on the plasma membrane, and are internalized as dimers/oligomers. This study is the first to provide direct in vivo visualization of GPCR dimerization/oligomerization, during trafficking to and from the plasma membrane. (C) 2017 Elsevier B.V. All rights reserved.
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