Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor

Abstract

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.

Figure 1. Transient expressions of epitope-tagged AT2 receptors.

(A) Schematic illustration of C-terminally or N-terminally tagged AT2 receptor variants. (B) Confocal images of transiently expressed epitope-tagged AT2 receptor variants. Plasmid (1 µg) was transiently transfected into cells (on a 25-mm coverslip). After 24 hr, cells that transfected with AT2-GFP were examined by live cell imaging; cells that transfected with Myc-AT2, HA-AT2 or AT2-FLAG were fixed, permeabilized and probed with an epitope-specific antibody as described in Methods. Following incubation with an FITC-conjugated secondary antibody, fluorescent images of transiently transfected cells were captured using a 63× water immersion objective with a Leica SP5 confocal microscope. (C) Pull-down immunoblot protein analysis of HEK293 cells that were transiently transfected with Myc-AT2. The Myc-AT2 expression construct or control empty vector (5 µg) was transiently transfected into HEK293 cells (in a 100-mm dish) as described in Methods. After 48 hr, cells were lysed, immunoprecipitated and probed with an anti-Myc antibody. Specific immunoreactive protein bands are indicated with asterisks. Ab V_H: Antibody heavy chain. Data shown is a representative of 2–3 independent experiments with similar results.

Figure 2. Stable expressions of epitope-tagged AT2 receptors.

(A) Confocal images of stably expressed C-terminally or N-terminally epitope-tagged AT2 receptor variants. Stably transfected cells were seeded and grown on coverslip for two days. Following fixation and permeabilization, the cells were probed with epitope-specific antibody and then incubated with an FITC-conjugated secondary antibody as described in Methods. Fluorescent images were captured using a 63× water immersion objective with a Leica SP5 confocal microscope. Pull-down immunoblot protein analysis of cells stably transfected with N-terminally tagged (B) or C-terminally tagged (C) AT2 receptor variants. To increase surface expression of AT2, CHO-K1 and PC12 cells that stably expressed Myc-AT2 were serum-starved overnight. Stably transfected cells or respective controls were lysed in RIPA buffer, immunoprecipitated and probed with an anti-Myc (B), anti-GFP or anti-FLAG (C) antibody as indicated. Cells stably transfected with corresponding empty vector were used as controls. Specific immunoreactive protein bands are indicated with asterisks. Ab V_H: Antibody heavy chain. Data shown is a representative of 2–3 independent experiments with similar results.

Figure 3. Roles of post-translational processing on cell surface expression of AT2 receptors.

(A) Western protein blot analysis of the membrane fraction of cells that stably transfected with epitope-tagged AT2 receptor variants. Cells that stably expressed Myc-AT2 were cultured in 100 mm dish until confluence. Cells were then cultured in serum-free medium overnight the day before cell lysis. Membrane fraction was prepared as described in Methods. Protein blot was probed with an anti-GFP or an anti-Myc antibody as indicated. (B) Cell surface immunoprecipitation of Myc-AT2. Cells were cultured until confluence and then incubated with serum-free medium overnight the day before cell lysis. Cell surface AT2 receptors were immunoprecipitated as described in Methods. Immunoprecipitated proteins were separated in 10% SDS-PAGE and protein blot was probed with an anti-Myc antibody. (C) Glycosylation on epitope-tagged AT2 receptor variants. HEK293 cells (100 mm disk with >90% confluence) that stably expressed Myc-AT2 or AT2-GFP were treated with or without 1 µg/ml tunicamycin for 24 hr. Cells were then lysed in RIPA buffer, epitope-tagged AT2 is immunoprecipitated, and protein blot was probed with an anti-Myc or an anti-GFP antibody as indicated. (D) Dimer and oligomer formation of AT2 receptors. The AT2-GFP expression construct (5 µg) was transiently co-transfected with Myc-AT2 expression construct (5 µg) or empty vector controls into HEK293 cells. Two days later, cells were lysed in the RIPA buffer. The dimeric or oligomeric AT2 receptors were immunoprecipitated with a polyclonal anti-GFP antibody and detected with a monoclonal anti-Myc antibody. Cells stably transfected with corresponding empty vector were used as controls. Specific immunoreactive protein bands are indicated with asterisks. Ab V_H: Antibody heavy chain. Data shown is a representative of 2–4 independent experiments with similar results.

Figure 4. Ligand-independent constitutive activities of epitope-tagged AT2 receptors.

(A) Serum starvation-induced DNA fragmentation. Stably transfected CHO cells (5×10⁵), PC12 cells (5×10⁵), and HEK cells (1×10⁶) were cultured in serum-free medium for 72 hr. Genomic DNA was extracted and fractionated on 2% TAE agarose gel as described in Methods. (B) Cell proliferation analysis. Stably transfected CHO cells (1×10⁵ cells), PC12 cells (5×10⁵ cells) and HEK293 cells (5×10⁵ cells) were seeded on 60-mm dishes and cultured for 5 days. (C) Stably transfected HEK293 cells (5×10⁵ cells on 60 mm dish) were cultured for 5 days in the absence or presence of 1 µM ANGII. (D) Stably transfected CHO-K1 (1×10⁵ cells on 60 mm dish) and HEK293 cells (5×10⁵ cells on 60 mm dish) were cultured for 5 days in the presence of 1 µM ANGII or 10 µM PD 123319, respectively. Cell numbers were counted using Beckman coulter Vi-cell TM XR cell viability analyzer. Cell proliferation were expressed as fold change of cell number. Data shown is mean ± SEM from 4∼6 independent experiments. *indicates P<0.05.

Figure 5. Ubiquitination of transiently expressed epitope-tagged AT2 receptors.

(A, B) The HA-ubiquitin expression construct (5 µg) were transiently co-transfected with Myc-AT2 expression construct or control empty vector (5 µg) into HEK293 cells (in a 100-mm dish); or HA-ubiquitin expression construct (5 µg) was transiently transfected into HEK293 cells (in 100-mm dish) that stably expressed Myc-AT2 or control HEK293 cells that stably transfected with empty vector. After 48 hr, cells were incubated with 10 µg/ml MG132 for 5 hr and then lysed in 1 ml of RIPA buffer containing 20 mM N-ethylmaleimide. Cell lysate was subject to immunoprecipitation with an anti-Myc antibody and then probed with an (A) anti-HA or (B) anti-Myc antibody. (C, D) Expression constructs of HA-ubiquitin (5 µg) and AT2-GFP (5 µg) were transiently co-transfected into HEK293 cells (in 100 mm dish); or HA-ubiquitin expression construct (5 µg) was transiently transfected into HEK293 cells that stably expressed AT2-GFP (in a 100-mm dish). After 48 hr, cells were lysed in 1 ml of RIPA buffer. Cell lysate was subject to immunoprecipitation with an anti-GFP antibody and then probed with an (C) anti-HA or (D) anti-ubiquitin antibody. Ab V_H: Antibody heavy chain. Data shown is representative of 2–3 independent experiments with similar results.