Dimethylsulphoxide as a tool to increase functional expression of heterologously produced GPCRs in mammalian cells

High-level overexpression of G protein-coupled receptors GPCRs in mammalian cells remains a difficult task inspite of newly developed virus based expression systems. Here, we show that the functional expression level of the recombinant bradykinin receptor (B(2)R) in mammalian cells can be increased up to sixfold just by the addition of dimethylsulphoxide in the culture medium. Total expression level, cellular localization and binding affinity of the recombinant receptor for its endogenous ligand remains unaltered. The strategy presented here, with recombinant B(2)R as a case example, is applicable to other GPCRs and provides a generic tool to improve the functional expression level of recombinant GPCRs in mammalian cells.     

Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells

The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B(max) values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (Kd = 11 nM) as to MC4r expressed in mammalian cells. Functional coupling of the highest expressed C-terminal tagged receptor to endogenous Galpha protein was demonstrated through GTPgammaS binding upon agonist stimulation of the receptor. Ki values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells.     

Functional overexpression and characterization of human bradykinin subtype 2 receptor in insect cells using the baculovirus system

Bradykinin exerts its actions via binding to B1 and B2 receptors (B1R and B2R), which are members of G protein-coupled receptor superfamily. B2R is constitutively expressed in a variety of cells such as endothelial cells, vascular smooth muscle cells, and cardiomyocytes and it is an important drug target for the treatment of cardiovascular disorders. During this study, the human B2R was functionally overexpressed in insect cells using the baculovirus expression system. The maximum expression level in Sf9 cells under optimized condition was 10 pmol/mg. This corresponds to approximately 0.25 mg active receptor per liter culture. The recombinant receptor showed high affinity for its endogenous ligand bradykinin, similar to the B2R expressed in native tissues. Functional coupling of the recombinant receptor to the endogenous G alpha(s) protein was demonstrated via cAMP release assay upon agonist stimulation. Confocal laser scanning microscopy and immunogold-labeling experiment revealed that the recombinant B2R was mainly localized intracellularly and only a minor fraction of the recombinant receptor reached the plasma membrane. To our knowledge, this is the first report of high level expression of recombinant B2R in insect cells and provides a way for large scale production and structural characterization of this receptor.     

Vortex flow filtration of mammalian and insect cells

The use of vortex flow filtration for harvesting cells or conditioned medium from large scale bioreactors has proven to be an efficient, low shear method of cell concentration and conditioned medium clarification. Several 8–10 L batches of the human histiocytic lymphoma U-937 cell line (ATCC CRL 1593) were concentrated to less than 1 L by vortex flow filtration through a 3.0 m membrane. An aggressive filtration regimen caused a 17% loss of cell viability and a 32% loss of IL-4 receptor binding capacity when compared to a batch centrifuged control. A reduction of the rotor speed from 1500 to 500 RPM and reduction of system back pressure from 10 to 0 PSIG resulted in cell viability and IL-4 binding capacity comparable to the control. Several 10 L batches of baculovirus infected Sf-9 cells were also concentrated to less than 1 L by vortex flow filtration through a 3.0 m membrane. SDS-PAGE analysis of filtrate samples showed that aggressive filtration caused cell damage which led to contamination of the process stream by cellular lysate. When rotor speed was reduced to 500 RPM and system back pressure was reduced to 0 PSIG, the amount of contaminating lysate proteins in filtrate samples was comparable to a batch centrifuged control.     

Angiotensin II type 1 receptor blockade attenuates renal fibrogenesis in an immune-mediated nephritic kidney through counter-activation of angiotensin II type 2 receptor

The relative roles of angiotensin II (Ang II) type 1 receptor (AT(1)R) and Ang II type 2 receptor (AT(2)R) in immune-mediated nephritis are unknown, and the effect of the blockade of AT(1)R and its indirect counter-activation of AT(2)R relative to the anti-fibrotic action in this disease is unclear. To address this question, we studied the role of AT(1)R and AT(2)R in anti-glomerular basement membrane nephritis in SJL mice. Groups of mice were treated with either an AT(1)R antagonist (CGP-48933; CGP group), an AT(2)R antagonist (PD-123319; PD group), both (CGP/PD group), or a vehicle (PCt group) from Day 29 to 56. At Day 56 post-treatment, fibrosis-related parameters such as interstitial matrix deposition, and the expression of genes of TGF-beta1, plasminogen activator inhibitor-1, and type I collagen were significantly reduced in the kidney in the CGP group. There were no significant effects on these parameters in the PD group. However, this anti-fibrotic action by CGP-48933 was totally abolished by co-treatment with PD-123319 in the CGP/PD group. The gene expression of renin was significantly increased in the kidneys in the CGP and CGP/PD groups, suggesting that CGP-48933 had increased Ang II generation in those groups. In conclusion, counter-activation of AT(2)R by increased Ang II under AT(1)R blockade likely conferred an anti-fibrotic protection in this model.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Expression and purification of human (pro)renin receptor in insect cells using baculovirus expression system

The renin-angiotensin (RA) system is important for the regulation of blood pressure and electrolyte balance, and renin is the rate-limiting enzyme in this system. The recent discovery of (pro)renin receptor (PRR) has reinforced the functional role of the RA system. PRR non-proteolytically activates prorenin and its role has attracted the attention of researchers towards the RA system. However, there is insufficient information on the biochemical structure and biological functioning of PRR due to the difficulty of measuring PRR expression. In this work, human PRR (hPRR) with intact transmembrane and C-terminal domain (hPRR-wTM) and PRR without this domain (hPRR-w/oTM) were expressed in insect cells using baculovirus expression system (BES). Both hPRR-wTM and hPRR-w/oTM were fused with FLAG peptide by its N-terminus. Most of the hPRR-wTM was expressed in cell fraction and hPRR-w/oTM was secreted into the culture medium. hPRR-wTM was solubilized from the membrane fraction of recombinant baculovirus-infected cells by various detergents, suggesting that hPRR-wTM might be a transmembrane protein. hPRR-wTM was purified from the solubilized fraction using anti-FLAG M2 antibody agarose. Binding of purified hPRR-wTM to renin immobilized onto sensor chips was directly proportional to the hPRR-wTM concentration. Approximately 225 μg of functional hPRR-wTM was purified from 80 ml of baculovirus-infected cell culture. Scale-up of this system will lead to mass production and crystallization of hPRR-wTM and determination of its biochemical structure and biological function.     

Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells

Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca²⁺ ([Ca²⁺]_i) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1 in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca²⁺]_i elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca²⁺]_i chelator; KN-93, a Ca²⁺/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca²⁺]_i-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs.     

Mechanical stress-evoked but angiotensin II-independent activation of angiotensin II type 1 receptor induces cardiac hypertrophy through calcineurin pathway

Mechanical stress can induce cardiac hypertrophy through angiotensin II (AngII) type 1 (AT₁) receptor independently of AngII, however, the intracellular mechanisms remain largely indeterminate. Since calcineurin, a Ca²⁺-dependent phosphatase, plays a critical role in pressure overload-induced cardiac hypertrophy, we therefore, asked whether calcineurin is involved in the AT₁ receptor-mediated but AngII-independent cardiac hypertrophy. Mechanical stretch failed to elicit hypertrophic responses in COS7 cells co-transfected with plasmid of AT₁ receptor and siRNA of calcineurin. Mechanical stresses for 2 weeks in vivo and for 24 h in vitro significantly induced upregulation of calcineurin expression and hypertrophic responses, such as the increases in cardiomyocytes size and specific gene expressions, in cardiomyocytes of angiotensinogen gene knockout (ATG^−/−) mice, both of which were significantly suppressed by a specific calcineurin inhibitor FK506, suggesting a critical role of calcineurin in mechanical stress-induced cardiac hypertrophy in the ATG^−/− mice. Furthermore, an AT₁ receptor blocker Losartan not only attenuated cardiac hypertrophy but also abrogated upregulation of cardiac calcineurin expression induced by mechanical stresses in the AngII-lacking mice, indicating that calcineurin expression is regulated by AT₁ receptor without the involvement of AngII after mechanical stress. These findings collectively suggest that mechanical stress-evoked but AngII-independent activation of AT₁ receptor induces cardiac hypertrophy through calcineurin pathway.     

Ligand-supported homology modeling of the human angiotensin II type 1 (AT(1)) receptor: insights into the molecular determinants of telmisartan binding

Angiotensin II type 1 (AT(1)) receptor belongs to the super-family of G-protein-coupled receptors, and antagonists of the AT(1) receptor are effectively used in the treatment of hypertension. To understand the molecular interactions of these antagonists, such as losartan and telmisartan, with the AT(1) receptor, a homology model of the human AT(1) (hAT(1)) receptor with all connecting loops was constructed from the 2.6 A resolution crystal structure (PDB i.d., 1L9H) of bovine rhodopsin. The initial model generated by MODELLER was subjected to a stepwise ligand-supported model refinement. This protocol involved initial docking of non-peptide AT(1) antagonists in the putative binding site, followed by several rounds of iterative energy minimizations and molecular dynamics simulations. The final model was validated based on its correlation with several structure-activity relationships and site-directed mutagenesis data. The final model was also found to be in agreement with a previously reported AT(1) antagonist pharmacophore model. Docking studies were performed for a series of non-peptide AT(1) receptor antagonists in the active site of the final hAT(1) receptor model. The docking was able to identify key molecular interactions for all the AT(1) antagonists studied. Reasonable correlation was observed between the interaction energy values and the corresponding binding affinities of these ligands, providing further validation for the model. In addition, an extensive unrestrained molecular dynamics simulation showed that the docking-derived bound pose of telmisartan is energetically stable. Knowledge gained from the present studies can be used in structure-based drug design for developing novel ligands for the AT(1) receptor.     

Unique "delta lock" structure of telmisartan is involved in its strongest binding affinity to angiotensin II type 1 receptor

Angiotensin II type 1 receptor (AT1 receptor) blockers (ARBs) are one of the most popular anti-hypertensive agents. Control of blood pressure (BP) by ARBs is now a therapeutic target for the organ protection in patients with hypertension. Recent meta-analysis demonstrated the possibility that telmisartan was the strongest ARB for the reduction of BP in patients with essential hypertension. However, which molecular interactions of telmisartan with the AT1 receptor could explain its strongest BP lowering activity remains unclear. To address the issue, we constructed models for the interaction between commonly used ARBs and AT1 receptor and compared the docking model of telmisartan with that of other ARBs. Telmisartan has a unique binding mode to the AT1 receptor due to its distal benzimidazole portion. This unique portion could explain the highest molecular lipophilicity, the greatest volume distribution and the strongest binding affinity of telmisartan to AT1 receptor. Furthermore, telmisartan was found to firmly bind to the AT1 receptor through the unique “delta lock” structure. Our present study suggests that due to its “delta lock” structure, telmisartan may be superior to other ARBs in halting cardiovascular disease in patients with hypertension.     

Intronic enhancement of angiotensin II type 2 receptor transgene expression in vitro and in vivo

The angiotensin II type 2 receptor (AT2R) can influence a variety of intracellular signaling molecules and cellular functions. However, its physiological functions and the roles of introns in the regulation of its expression have not been well determined. We first demonstrated that both intron 1 and intron 2 of AT2R could significantly enhance AT2R overexpression. Thus, we have provided some new prerequisites for further studies on the mechanisms that control AT2R gene expression. Next, we established a highly efficient method of delivering this receptor in vitro and in vivo using an AT2R recombinant adenoviral vector containing two introns of the AT2R. The vector may be useful in helping to uncover AT2R physiological functions and also for gene therapy related to AT2R. Moreover, we determined the important role of introns in gene expression cassettes and the inconsistency of expression between the targeted gene and the reporter gene.     

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.     

The clinical efficacy of angiotensin II type1 receptor blockers on inflammatory markers in patients with hypertension: a multicenter randomized-controlled trial; MUSCAT-3 study

Purpose: The purpose of present study was to evaluate the clinical efficacy of irbesartan on the anti-inflammatory and anti-oxidative stress effect in patients with hypertension compared to other ARBs. Further, we assessed the effect of the ARBs on kidney function and urinary albumin excretion.

Methods: Eighty-five outpatients with hypertension who took an ARB except irbesartan more than 3?months were assigned into two groups, one continued the same ARB and the other switched the ARB to irbesartan for 6?months.

Results: Although blood pressures were equally controlled (continue group: 148?±?2/79?±?2?mmHg to 131?±?2/74?±?2?mmHg; switch group: 152?±?2/81?±?2?mmHg to 132?±?2/74?±?2?mmHg; p?<?0.001 each), the inflammatory markers (hsCRP, PTX3, MCP-1) and oxidative stress marker (MDA-LDL) did not change after 6?months in both groups. Urinary albumin excretion was significantly reduced only in the switch group without renal function deterioration (switch group 292.4?±?857.9?mg/gCr to 250.6?±?906.5?mg/gCr, p?=?0.012).

Conclusion: These results provide knowledge of the characteristics of irbesartan, suggesting appropriate choice of ARBs in the treatment for hypertension should be considered.     

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text]     

Expression and purification of non-glycosylated Trypanosoma brucei transferrin receptor in insect cells

The transferrin receptor of the parasite Trypanosoma brucei is a heterodimeric protein complex encoded by the 2 expression site-associated genes (ESAGs) 6 and 7. ESAG6 is a heterogeneously glycosylated protein of 50-60 kDa modified by a glycosylphosphatidylinositol anchor at the C-terminus, while ESAG7 is a 40-42 kDa glycoprotein carrying an unmodified C-terminus. In order to determine whether glycosylation is necessary for dimer formation and ligand binding, the receptor was expressed in insect cells in the presence of tunicamycin. When insect cells were infected with recombinant ESAG6/ESAG7 double expressor baculovirus and grown in the presence of tunicamycin, non-glycosylated forms of ESAG6 and ESAG7 of 46 and 36 kDa, respectively, were synthesized. The non-glycosylated ESAG6 and ESAG7 were capable of forming a heterodimer and of binding transferrin. This results shows that glycosylation is not necessary for synthesis of a functional T. brucei transferrin receptor.