Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.
Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.
School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Lili
L
Jiang
Gladys M K
GM
Teng
Elaine Y M
EY
Chan
Shannon W N
SW
Au
Helen
H
Wise
Susanna S T
SS
Lee
Wing-Tai
WT
Cheung
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008970:Molecular Weight; D016716:PC12 Cells; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D051381:Rats; D044139:Receptor, Angiotensin, Type 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D054875:Ubiquitination
D000939:Epitopes; D044139:Receptor, Angiotensin, Type 2