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.
Shukla
Arun Kumar
AK
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Street 3, 60438 Frankfurt/M, Germany. arun.shukla@meibp-frankfurt.mpg.de
Haase
Winfried
W
Reinhart
Christoph
C
Michel
Hartmut
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007313:Insecta; D043782:Receptor, Bradykinin B2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D043782:Receptor, Bradykinin B2; D011994:Recombinant Proteins; D000242:Cyclic AMP