Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 overexpression induces beta-2 adrenergic receptor desensitization in airway smooth muscle cells

Background

Beta-2 adrenergic receptor (β₂AR) gene polymorphisms are critical to the response of asthma patients to both short- and long-acting β₂AR agonists, the most effective class of bronchodilators and a mainstay of asthma management (1). Worldwide prevalence of adult asthma has been reported at 4.3%, though more than 21-times this number have been reported in some countries and increasing trends have been reported with urbanization (2). In asthma patients, β₂AR downregulation is critical to the action of asthma rescue treatments based on β₂AR agonists (3). However, multi-mechanism hormonal desensitization can result in tolerance, or bronchodilator desensitization, leading to rescue treatment failure (3). Thus, there is an urgent need to better understand the complex mechanisms involved in bronchodilator desensitization in order to develop preventative strategies.

Short-acting β₂AR agonists are life-saving rescue agents, though regular use is not recommended due to safety and tolerance-development concerns (4). Similar concerns pertaining to long-acting β₂AR agonists have restricted these medications to patients with asthma that is not acceptably controlled by inhaled corticosteroids (5). β₂AR agonists predominantly function through β₂AR downregulation, characterized by the sustained activation of G protein (guanylate binding protein)-coupled receptors, thus resulting in a time-dependent reduction of receptor density in intact cells (3). Frequent β₂AR downregulation, however, may contribute to β₂AR desensitization through mechanisms such as receptor phosphorylation, G-protein uncoupling, receptor internalization, decreased receptor mRNA and protein synthesis, and increased receptor degradation (6).

The mechanistic process of β₂AR desensitization involves initial rapid desensitization followed by heterologous and homologous desensitization (6). Homologous desensitization is mediated by G protein-coupled receptor kinases (GRKs) with specific roles in ligand binding (7). Furthermore, considerable evidence indicates that carboxyl-terminal serine 355, 356, and 364 may play roles in GRK-mediated phosphorylation, thus contributing to β₂AR desensitization (7,8). Most contemporary research agrees that G protein-coupled receptor kinase 2 (GRK₂) is responsible the majority of agonist-dependent receptor phosphorylation, central to β₂AR desensitization (9).

Agonist stimulation of the β₂ARs has been reported to lead to both ubiquitination and lysosomal degradation, central features of β₂AR desensitization (10). In asthmatic and β₂AR desensitization mouse models, the levels of inflammatory cells, bronchoalveolar lavage fluid (BALF) cytokines, serum immunoglobulin E (IgE), and, most notably, the protein target of ubiquitin, Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI₂), have been reported to be statistically increased in mice exhibiting β₂AR desensitization in recent comparative proteomics studies (11-13). RhoGDI₂ is an important regulating factor of Rho (Ras homologue). Additionally, lysosomal degradation leading to progressive β₂AR desensitization is regulated by Rab11, which impacts recycling and lysosome targeting of β₂-adrenergic receptors, and Rab11 binding is determined, in part, by Arg(333) and Lys(348) (14). Because both ubiquitination and lysosomal degradation are central mechanisms in the development of β₂AR desensitization (10), the role of RhoGDI₂ in β₂AR desensitization merits further exploration. As the regulatory mechanism we determine to study which is relevant to Rho, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are both regulatory factor of Rho, so RhoGEF and RhoGAP may play an important part in the process. RhoGEF can increase the activity of Rho protein and RhoGAP down-regulates activity of Rho protein.

The current study employs airway smooth muscle cells (ASMCs) from mice transfected with lentivirus vector-mediated RhoGDI₂ overexpression to explore the effect of RhoGDI₂ overexpression on β₂AR desensitization. Furthermore, mRNA and protein expressions of RhoGDI₂, β₂AR, GEF, GAP, and GRK₂ were explored to provide a basis for determination of the relationship between these proteins and β₂AR desensitization.

Materials and methods

Animal subjects

Female BALB/c mice aged 6-8 weeks (mean body weight 20±2 g) were purchased from the Laboratory Animal Center of Nantong University (Nantong, China). All mice were housed in temperature-controlled cages at 20-25 °C with a 12-hour dark/light cycle and with ad libitum access to food and water for one week prior to the experiments. BALB/c mice ASMCs from mice were grouped as untreated (control), GFP lentivirus-infected (negative control), and RhoGDI₂ vector-infected (RhoGDI₂ overexpression group) for these experiments. All animal protocols were approved by the Institutional Animal Care and Use Committee of Nantong University and conformed to the International Guidelines for Ethical Use of Animals.

Primary culture and purification of ASMCs

Mice ASMCs were primarily and serially cultured. ASMCs were purified by differential adherence (15) and identified by appraisal α-actin protein immunocytochemical staining, as previously described (16,17). Briefly, the trachea and pulmonary tissues were collected from mice killed by neck-breaking and soaked in 75% alcohol for 2 min. Hyaline tissues were attained by removing tracheal ectoblasts, cutting the trachea lengthways with ophthalmic tweezers, and scraping the inner membrane gently with scalper. Resultant hyaline tissues (<1 mm³) were affixed equidistantly with dental probe in the bottom of a 25 mL culture bottle. Cells were cultured in covered, inverted culture bottles with 2 mL high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, USA) containing 20% fetal bovine serum (FBS) (GIBCO, USA) at 37 °C, in a 5% CO₂ incubator for 3 hours without agitation, in which cells did not touch medium. Upon attaining dry tissues, bottles were inverted to submerge cells in the medium, cultured for 3 days with loose covering, and mixed with 5 mL of new medium on day 3. All medium was replaced on day 6, and thereafter half the medium was changed every 5 days. When outer cell boundaries were fused >80% under inverted phase contrast microscope (Olympus, Tokyo, Japan), medium was removed, cells were washed D-Hanks solution 3×, 2 mL of 0.25% trypsin was added with mild agitation to ensure even distribution, and cells were cultured for 5 min, producing bright appearance and retracted cytoplasm under microscopic examination. Bottles were briefly agitated, and 6 mL of high-glucose DMEM containing 20% FBS was added to stop digestion. Cells were dried repeatedly with Tubularis^TM (Biomics biotechnologies, China) and then inoculated at 1:1, 1:2, or 1:3 densities. Half of the medium was changed at 5-day intervals.

For purification, cells were digested with 0.25% trypsin, and DMEM containing 20% FBS was added for 15 min. Non-adherent cells were separated and subcultured for 15 min, which was repeated for twice, thereby using the smaller adherence time of fibroblasts (10-30 min adherence) compared to smooth muscle cells (1-4 h adherence) to separate and purify ASMCs (15).

Immunocytochemical staining

SABC-FITC immunocytochemical staining was conducted using an immunohistochemical kit (Wuhan Boster Biotechnology Ltd., China), according to the instructions provided by the manufacturer. Briefly, cells cultured to 80% fusion were fixed with 4% paraformaldehyde for 20 min, washed with distilled water 3× for 3 min, subjected to 0.6% hydrogen peroxide for 30 min and 0.01 mol/L phosphate buffered saline (PBS) solution 3× for 2 min, treated with normal goat serum at room temperature for 20 min without washing, and treated with mouse Anti-α-actin monoclonal antibody (Wuhan Boster Biotechnology Ltd., China) for 20 min. Slides were then washed with 0.01 mol/L PBS 3× for 2 min, treated with biotinylation Goat Anti-Rabbit IgG (Santa cruz biotechnology, USA) for 20 min, washed with 0.01 mol/L PBS 3× for 2 min, treated with SABC-FITC for 20 min, and washed with 0.01 mol/L PBS for 3× for 5 min. Cells were then observed and photographed under a laser scanning microscope (Olympus, Tokyo, Japan).

Production of RhoGDI₂ recombinant lentivirus vectors

The RhoGDI₂ gene (NCBI NM_001175.4) coding sequence was amplified by PCR and subcloned into a lentivirus expression plasmid pWPXL-eGFP vector (TronoLab, France) along with BamH I and Mlu I restriction sites to construct a lentivirus-based overexpression vector carrying the RhoGDI₂ sequence (pWPXL -eGFP-RhoGDI₂), confirmed by PCR and DNA sequencing. The lentivirus expression plasmid pWPXL-eGFP-RhoGDI₂ and two packaging plasmids psPAX2 and pMD2.G (TronoLab, France) were co-transfected into human embryonic kidney cells (HEK293T) in serum-free medium using Lipofectamine^TM 2000 (Invitrogen Co., Carlsbad, CA), according the manufacturer’s instructions. Additionally, a lentivirus expression plasmid without RhoGDI₂ gene and two packaging plasmids were co-transfected into 293T cells to construct a blank control lentivirus. At 8 h after transfection, medium was completed replaced. At 48 h after transfection, culture medium was collected and centrifuged at 4,000 ×g and 4 °C for 10 min. The supernatant was filtered (0.45 μm) into a Plus-20 centrifugal ultrafiltration (Biomics biotechnologies, China) and centrifuged at 4,000 ×g to obtain high-titer lentivirus.

Establishment of stable RhoGDI₂ overexpression ASMCs

Primary ASMC cultures were plated at a concentration of 1.5×10⁵ cells/well in six-well plates and infected with RhoGDI₂-GFP lentivirus vectors (RhoGDI₂ overexpression group), GFP lentivirus vectors (negative control group), or no infection (control) in serum-free medium for 12 h. Cells were then washed and embedded in complete medium for 3 days. GFP expression was examined by fluorescence microscopy (Olympus, Tokyo, Japan). 4 days after infection, ASMCs were used to the subsequent experiments.

Real-time RT-PCR detection of RhoGDI₂, β₂AR, GEF, GAP, and GRK₂ mRNA expressions

Total RNA was extracted from ASMCs with Trizol reagent (Invitrogen, USA). The ratio of absorbance at 260 and 280 nm (A_260/280) was used to assess RNA purity. cDNA synthesis was performed using oligo (dT) primer (Sangon BiotechCo., Ltd, Shanghai, China) and M-MLV reverse transcriptase (Promega, USA). Quantitative real-time PCR detection was performed using a SYBR green real-time PCR kit (Takara, Japan) in real-time PCR system (BioRad, USA). Housekeeping gene α-tubulin expression was quantified and used for normalization. All primers were designed using Primer Premier 5 (PREMIER Biosoft International, Palo Alto, California, USA) (Table 1). Thermal cycling was conducted for 5 min at 95 °C (pre-denaturation), followed by 25 cycles at 95 °C for 20 s (denaturation), 55 °C for 30 s (annealing), and 72 °C for 30 s (stretching), and 72 °C for 7 min (final stretching). 2^–ΔΔCT was used to calculate relative changes in gene expression from qRT-PCR (ΔCT = CT_{target gene}–CT_α-tubulin, ΔΔCT = ΔCT _experimental –ΔCT _control).

Table 1. Primer sequences for real-time RT-PCR.
Full Table

Western blot detection of RhoGDI₂, β₂AR, GEF, GAP, and GRK₂ protein expressions

ASMCs were washed 2× with cold PBS, treated with 2× lysis buffer comprised of 100 mmol/L Tris-HCl (pH=6.8), 20 g/L mercaptoethanol, 200 g/L glycerol, and 40 g/L SDS, and lysed on ice for 10 min. Cells were homogenized by ultrasonication, and centrifuged at 4 °C at 12,000 ×g for 15 min. Protein concentration was measured by trace protein nucleic acid analyzer (Implen, Germany). Protein samples (20 ug) were subjected to 8% SDS-PAGE and transferred onto poly (vinyl idene fluoride) (PVDF) membranes (Pall, USA) treated with tris-buffered saline and tween-20 solution (TBST) containing 50 g/L skim milk at room temperature for 1 h. Then, rabbit anti-mouse RhoGDI₂ (FL-201) polyclonal antibody (sc-11359), rabbit anti-mouse β₂AR (H-73) polyclonal antibody (sc-9042), rabbit anti-mouse RhoGEF [p115/Lsc (H-165)] polyclonal antibody (sc-20804), rabbit anti-mouse ARHGAP[22/24 (H-162)] polyclonal antibody (sc-99112), rabbit anti-mouse GRK₂ (C-15) polyclonal antibody (sc-562), and rabbit anti-mouse a-tubulin polyclonal antibody (sc-135659) (Santa Cruz Biotechnology, CA, USA) were added and incubated at room temperature for 2 h. Membranes were washed 3× with TBST and incubated with diluted horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, CA, USA) at room temperature for 2 h. Membranes were washed 3× with TBST, and bands were detected using an ECL + plus^TM kit (Beyotime Ltd., China) The relative protein expressions were normalized to a-tubulin.

Radioligand receptor binding assay

ASMCs were centrifuged at 250 ×g at 4 °C for 10 min, and the resultant pellet was resuspended in 8 volumes of hypotonic buffer at 0 °C, allowed to swell for 10 min, and centrifuged at 12,000 ×g at 4 °C for 15 min. When 90% of cells were broken, as determined by phase-contrast microscopy, the pellet was resuspended in binding buffer and homogenized with a Polytron homogenizer (Kinematica, Basel, Switzerland). ASMCs were incubated with (125I) iodocyanopindolol (ICYP) (sp act: 7,400×1,010 Bq/mmol; 3-100 pmol/L) (GE Healthcare Life Sciences, USA) in the presence or absence of excess Iso (Nuclear Medicine, Shanghai University of Traditional Chinese Medicine, China) (200 μmol/L) in 25 mmol/L Tris-HCl buffer (pH 7.4) containing 154 mmol/L NaCl and 1.1 mmol/L ascorbic acid to prevent oxidation of Iso, in a final volume of 250 μL. β₁AR density was analyzed by ICYP saturation binding in the presence of 0.1 μmol/L ICI 118551 (Nuclear Medicine, Shanghai University of Traditional Chinese Medicine, China), a β₂-selective antagonist. At this concentration, virtually all β₂-receptors were occupied. β₂AR density was analyzed by ICYP saturation binding in the presence of 0.1 μmol/L CGP 20712 A (Nuclear Medicine, Shanghai University of Traditional Chinese Medicine, China), a β₁-selective antagonist (18). Cells were incubated in triplicate at 37 °C for 120 min for optimal specific binding, and terminated by rapid filtration through GF/C glass-fiber filters (Whatman Inc., Clifton, NJ, USA). Each filter was rapidly washed with 3×5 mL ice-cold 25 mmol/L Tris-HCl buffer (pH 7.4) and counted in the Auto Gamma Counting System (PerkinElmer, USA) at an efficiency of 80%. Specific binding was calculated by subtracting nonspecific binding from total binding. Protein concentration was determined by Lowry method with bovine serum albumin as a standard.

Statistical analysis

All data were reported as means ± standard deviation (SD), and statistical analyzed by SPSS vs.17.0 (SPSS, Inc, Chicago, USA). Normally distributed and homogenous data sets were compared by one way ANVOA and the least significant difference method (LSD) as post hoc test. P-values of less than 0.05 were considered statistically significant (P<0.05).

Results

ASMC growth

Primarily cultured ASMCs demonstrated typical characteristics, including a peak and valley growth trend (Figure 1A). Furthermore, specific smooth muscle α-actin filaments were observed parallel to the longitudinal axis in the cytoplasm (Figure 1B). The percentage of positive expression of specific smooth muscle α-actin filament was more than 95%.

Figure 1. Typical airway smooth muscle cells (ASMCs). (A) Third generation ASMCs showing typical peak and valley formations under inverted phase contrast microscope (×100); (B) ASMCs positive for smooth muscle α-actin filaments (green: FITC) under fluorescence microscopy (×200).

Stable overexpression of RhoGDI₂ in ASMCs

The recombinant lentivirus plasmid pWPXL-RhoGDI₂ was successfully constructed, and stablely transfected into ASMCs in the RhoGDI₂ overexpression group expressing GFP were observed under a fluorescence microscope (Figure 2). Fluorescence microscopy indicated infection efficiency >80%. Real-time RT-PCR demonstrated that the mRNA expression of RhoGDI₂ in the RhoGDI₂ overexpression group was about 377-fold higher than that in the control group (Figure 3A). Furthermore, western blot indicated that the protein expression of RhoGDI₂ was significantly higher in the RhoGDI₂ overexpression group than those in other groups (both P<0.05). The protein expression of RhoGDI₂ in the RhoGDI₂ overexpression group was approximately 3-fold higher than that observed in the control group (Figure 3B). There were no statistically significant difference in the mRNA and protein expressions of RhoGDI₂ between the negative control group and the control group (both P>0.05).

Figure 2. ASMCs displaying green fluorescence (GFP). ASMCs in the negative control group in visible light (A) (×100) and fluorescence (B) (×100) microscope, respectively. ASMCs in RhoGDI₂ overexpression group in visible light (C) (×100) and fluorescence (D) (×200) microscope, respectively. Negative control group: ASMCs transfected with GFP lentivirus; RhoGDI₂ overexpression group: ASMCs transfected with lentivirus-mediated RhoGDI₂ overexpression.

Figure 3. RhoGDI₂ mRNA and protein expression levels in ASMCs. (A) 4 days after transfection, total RNA of ASMCs was extracted and assayed for RhoGDI₂ mRNA by real-time RT-PCR. α-tubulin was used as an internal control; (B1,2) 4 days after transfection, total protein of ASMCs was extracted and assayed for RhoGDI₂ protein by western blot. α-tubulin was used as an internal control. The data represents the means ± standard deviation (SD) of three independent experiments. *P<0.05 vs. control group; ^#P<0.05 vs. negative control group. Control group, the untreated ASMCs; Negative control group, ASMCs transfected with GFP lentivirus; RhoGDI₂ overexpression group, ASMCs transfected with lentivirus-mediated RhoGDI₂ overexpression.

The effects of RhoGDI₂ overexpression on mRNA and protein expressions of β₂AR, GEF, GAP and GRK₂ in ASMCs

The mRNA expression of β₂AR was significantly lower in the RhoGDI₂ overexpression group than those in other groups (both P<0.05). The mRNA expressions of GEF and GRK₂ were significantly increased along with increasing RhoGDI₂ expression, demonstrating a positive relationship (all P<0.05). Conversely, the mRNA expression of GAP significantly decreased with increasing RhoGDI₂, demonstrating a negative relationship (all P<0.05) (Figure 4A). Similar trends were observed in protein expressions, with RhoGDI₂ overexpression associated with significantly increased GEF and GRK₂ protein expressions while GAP and β₂AR protein expressions were significantly decreased (all P<0.05) (Figure 4B).

Figure 4. The effects of RhoGDI₂ overexpression on mRNA and protein expressions of β₂AR, GEF, GAP and GRK₂ in ASMCs. (A) 4 days after transfection, total RNA of ASMCs was extracted and assayed for β₂AR, GEF, GAP and GRK₂ mRNA by real-time RT-PCR. α-tubulin was used as an internal control; (B1,2) 4 days after transfection, total protein of ASMCs was extracted and assayed for β₂AR, GEF, GAP and GRK₂ protein by western blot. α-tubulin was used as an internal control. The data represents the means ± SD of three independent experiments. *P<0.05 vs. control group; ^#P<0.05 vs. negative control group.

The effects of RhoGDI2 overexpression on β₂AR density in ASMCs

β₂AR density was significantly lower in the RhoGDI₂ overexpression group than those in other groups (both P<0.05), with control and negative control groups exhibiting β₂AR density more than 2-fold higher than that observed in the RhoGDI₂ overexpression group (Figure 5). No significant difference was observed between the control group and negative control group in terms of β₂AR density (P>0.05).

Figure 5. The effects of RhoGDI₂ overexpression on β₂AR density in ASMCs. 4 days after transfection, β₂AR density was determined by radioligand receptor binding assay. The data represents the means ± SD of three independent experiments. *P<0.05 vs. control group; ^#P<0.05 vs. negative control group.

Discussion

In mouse ASMCs transfected with lentivirus vector-mediated overexpression of RhoGDI₂, mRNA and protein expressions of RhoGDI₂, GEF, and GRK₂ were significantly increased while β₂AR and GAP expression were significantly decreased. According to our experimental results, we draw the Schematic diagram for the regulation of β₂AR by RhoGDI2 (Figure 6). Furthermore, β₂AR density in RhoGDI₂ overexpression group was less than half as high in the control group, indicating that RhoGDI₂ may contribute to the reduced β₂AR density observed in cellular β₂AR desensitization. Thus, the role of RhoGDI₂ in reducing β₂AR density is an important contributing factor in the complex mechanism associated with bronchodilator tolerance in asthma patients, with these findings meriting further investigation in humans.

Figure 6. Schematic diagram for regulation of β₂AR by RhoGDI₂. [1]. A signal localizes the complex proximal to a membrane compartment; [2]. RhoGDI_2, GEF and GAP regulate Rho; [3]. Exchange of RhoGDP for RhoGTP; [4]. And allows the GTPase to associate with GRK₂; [5]. GRK₂ mediates homologous desensitization.

The mechanism of RhoGDIs action has been previously reported, with the molecule reported to act primarily as a down-regulator of Rho family GTPases and thus prevent nucleotide exchange and membrane association (19). Cytoplasmic RhoGDIs inactivate GTPases by combining with GDP Rho proteins, stranding them in the cytoplasm and inhibiting GDP dissociation from Rho proteins; furthermore RhoGDIs terminate signal transduction and regulate negative feedback (19). Recent evidence has also suggested that RhoGDIs may be positive regulators of Rho activities (20). Consist with previous findings that asthmatic mice exhibit β₂AR desensitization verified by inflammatory cell count, cytokine concentration of BALF, and serum IgE level when exposed to overexpression of RhoGDI₂ (11), the current study supports that RhoGDI₂ influences β₂AR desensitization. If RhoGDI₂ plays a negative regulatory role in β₂AR desensitization process, RhoGDI₂ may hypothetically be able to inhibit Rho protein activity, though this hypothesis requires further exploration. In previous studies of heart failure, it has been reported that inhibition of GRK₂ can attenuate β₂AR desensitization (21). Furthermore, these findings in heart failure have been linked with G-protein β₂AR signaling, resulting in maladaptive remodeling and treatment failure (22). Consistent with these findings, the current results indicate that as GRK₂ decreases, β₂AR also decreases, which may indicate that RhoGDI₂ acts as a regulator of β₂AR important in the β₂AR desensitization process.

The current study indicates that RhoGDIs are important regulators of GEF and GAP activities. Previously, it has been reported that GEF can increase the activity of Rho protein, thus accelerating hydrolyzation of GTP in a similar manner as GRKs (23). Furthermore, RhoGEFs catalyzes the release of GDP and combined GTP, thereby activating the RhoGTP enzyme, and RhoGAPs down-regulate Rho protein activity, thereby increasing the activity of GTPase (23). Notably, the current RT-PCR and western blot studies consistently indicated that GAP expression decreased as RhoGDI₂ increased, suggesting that RhoGDI₂ may inhibit GAP activity. These findings are consistent with previous reports in metastatic cancer indicating that RhoGDI2 metastasis inhibition works through Rho GTPases and potentially influence GAP hydrolysis in a mechanism distinct from membrane association inhibition (24).

RhoGDI₂ has been reported to mediate GRK₂ through the Rho protein, but the effect RhoGDI₂ has on GRK₂ is remains uncharacterized (25). In previous studies in genetically modified mice, GRK₂ has been linked with abnormal airway and cardiac responses (26), further indicating the potential importance of GRK₂ during the treatment response of asthma patients. Notably, the expression of RhoGDI₂ in the current study was found to positively correlate with that of GRK₂. Thus, RhoGDI₂ likely plays an important role in increasing the activity of GRK₂, performing a positive role in β₂AR sensitization; however, further studies of the exact, direct role of Rho will be required to fully determine this mechanistic pathway. Hypothetically, the action of RhoGDI₂ through the phosphorylation of its acceptor (7-9) suggests that it may also play a role in upstream intra-cellular signaling pathways relevant to bronchodilator desensitization. However, it remains likely that other, yet undetermined, intimidate factors exist.

In the current study, only indirect comparison of RhoGDI₂ and Rho expression using both mRNA and protein level are possible, a central limitation of the current experiments. Thus, further experiments will be required to fully elucidate the role of Rho in β₂AR desensitization. The full utility of lentivirus-based vector systems as clinical alternatives has not been fully investigates, necessitating extensive further work before these results can be translated into meaningful clinical interventions for β₂AR desensitization. Furthermore, the current data as well as data from subsequent in vivo and in vitro studies should be more carefully statistically analyzed by correlation and regression analysis to potentially indicate or conform the trends observed in the present study.

Conclusions

In conclusion, lentivirus vector-mediated overexpression of RhoGDI₂ in mice ASMCs was associated with lower expressions of β₂AR and GAP, while higher expressions of GEF and GRK₂, parallel to reduce the β₂AR density, potentially contributing to β₂AR desensitization. Thus RhoGDI₂ may be an important mediator of β₂AR desensitization in asthmatic patients. Extensive further research, however, will be required to elucidate the full role of RhoGDI₂ and other intermediates involved in β₂AR desensitization prior to the development of future in vivo targets and clinical treatments.

Acknowledgements

This study was supported by Natural Science Foundation of China (NO: 30971306), Six big talent peak in Jiangsu province project (the seventh batch NO: 033), Nantong social development project (NO: S2009023) and Nantong fourth period “226 high-level personnel training project” project.

Disclosure: The authors declare no conflict of interest.

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