Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system.
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 G alpha(q) 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.
Shukla
Arun Kumar
AK
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, D-60438 Frankfurt/Main, Germany.
Haase
Winfred
W
Reinhart
Christoph
C
Michel
Hartmut
H
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D007295:Inositol Phosphates / Q000378:metabolism; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D043782:Receptor, Bradykinin B2 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*
D007295:Inositol Phosphates; D043782:Receptor, Bradykinin B2; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D020928:Mitogen-Activated Protein Kinases; D001920:Bradykinin