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

✍ Scribed by Arun Kumar Shukla; Winfried Haase; Christoph Reinhart; Hartmut Michel

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 302 KB
• Volume: 99
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.20976

✦ Synopsis

Abstract

Bradykinin exerts its actions via binding to B~1~ and B~2~ receptors (B~1~R and B~2~R), which are members of G protein‐coupled receptor superfamily. B~2~R 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 B~2~R 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 B~2~R expressed in native tissues. Functional coupling of the recombinant receptor to the endogenous Gα~s~ protein was demonstrated via cAMP release assay upon agonist stimulation. Confocal laser scanning microscopy and immunogold‐labeling experiment revealed that the recombinant B~2~R 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 B~2~R in insect cells and provides a way for large scale production and structural characterization of this receptor. J. Cell. Biochem. 99: 868–877, 2006. © 2006 Wiley‐Liss, Inc.