Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system
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Arun Kumar Shukla
Abstract
Bradykinin, a vasoactive peptide, plays a crucial role in many cardiovascular processes via activation of the bradykinin subtype 2 receptor (B2R). B2R, a member of the G protein-coupled receptor (GPCR) superfamily, is a potential drug target in the treatment of cardiovascular disorders, pain and inflammation. In this study, human B2R was expressed at high levels in baby hamster kidney (BHK) cells using Semliki Forest virus-based vectors. The recombinant receptor was produced as a fusion protein with affinity tags and an expression level of 11 pmol/mg (i.e., approx. 0.2 mg of active receptor per liter of culture) was obtained. Radioligand binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (Kd=0.12 nM) and its pharmacological profile was similar to that of B2R in native tissues. Bradykinin-stimulated accumulation of inositol phosphate was observed in BHK cells expressing the recombinant receptor, which indicated the activation of endogenous Gαq protein by the recombinant B2R. Confocal laser scanning microscopy and immunogold staining revealed that the recombinant receptor was predominantly localized intracellularly. To the best of our knowledge, this is the first report of an affinity-tagged recombinant B2R been expressed at high levels in BHK cells and extensively characterized.
References
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Articles in the same Issue
- Protein aggregation in crowded environments
- Nitrite, a naturally occurring precursor of nitric oxide that acts like a ‘prodrug’
- Functional studies of the small subunit of EcoHK31I DNA methyltransferase
- Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase
- Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β recombinase, and identification of a folding intermediate
- Tyr-48, a conserved residue in ribotoxins, is involved in the RNA-degrading activity of α-sarcin
- Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects
- Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco
- Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide
- Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system
- A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57
- Subcellular localisation of human inositol 1,4,5-trisphosphate 3-kinase C: species-specific use of alternative export sites for nucleo-cytoplasmic shuttling indicates divergent roles of the catalytic and N-terminal domains
- The gating effect of calmodulin and calcium on the connexin50 hemichannel
- C-Terminal fusion of eGFP to the bradykinin B2 receptor strongly affects down-regulation but not receptor internalization or signaling
- Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII
- μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation
- Cathepsin L splice variants in human breast cell lines
