Format

Send to

Choose Destination
FASEB J. 2014 Sep;28(9):3965-74. doi: 10.1096/fj.14-252999. Epub 2014 Jun 2.

Visualization of the pH-dependent dynamic distribution of G2A in living cells.

Author information

1
Department of Chemistry and Biotechnology and State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
2
Department of Chemistry and Biotechnology and.
3
Department of Bioengineering, Graduate School of Engineering, and.
4
Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; and.
5
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
6
Department of Chemistry and Biotechnology and Department of Bioengineering, Graduate School of Engineering, and nagamune@bioeng.t.u-tokyo.ac.jp.

Abstract

G2A (from G2 accumulation) receptor is a member of the proton-sensing G-protein coupled receptor (GPCR) family and induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis, and immunity. The mechanism by which G2A-mediated signal transduction is regulated by the extracellular pH remains unresolved. Here, we first visualize the pH-dependent G2A distribution change in living cells by a sortase A-mediated pulse labeling technology: the short-peptide tag-fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye in the presence of lysophosphatidylcholine, and the labeled G2A was chased at acidic and neutral pHs in real time by microscope time course observations. G2A internalization from cell surfaces into intracellular compartments was observed to be inhibited under acidic pH conditions, and this inhibition was relieved at neutral pH. Additionally, the internalized G2A was redistributed onto cell surfaces by jumping from a neutral to an acidic pH. From quantitative image analysis data, we conclude the amount of G2A on the cell surface was controlled by suppressing the G2A internalization rate by one-tenth in response to the extracellular acidic pH, and this acidic pH-induced G2A accumulation on cell surfaces may be explained by proton-induced dissociation of G2A from endocytic machinery.

KEYWORDS:

GPCR; internalization; pH dependence; pulse labeling

PMID:
24891524
PMCID:
PMC5395726
DOI:
10.1096/fj.14-252999
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Atypon Icon for PubMed Central
Loading ...
Support Center