Winfried Haase

Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system.

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 (K d=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.

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 Gαs 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. J. Cell. Biochem. 99: 868–877, 2006.

Switching of the homooligomeric ATP-binding cassette transport complex MDL1 from post-translational mitochondrial import to endoplasmic reticulum insertion

The ATP‐binding cassette transporter MDL1 of Saccharomyces cerevisiae has been implicated in mitochondrial quality control, exporting degradation products of misassembled respiratory chain complexes. In the present study, we identified an unusually long leader sequence of 59u2003amino acids, which targets MDL1 to the inner mitochondrial membrane with its nucleotide‐binding domain oriented to the matrix. By contrast, MDL1 lacking this leader sequence is directed into the endoplasmic reticulum membrane with the nucleotide‐binding domain facing the cytosol. Remarkably, in both targeting routes, the ATP‐binding cassette transporter maintains its intrinsic properties of membrane insertion and assembly, leading to homooligomeric complexes with similar activities in ATP hydrolysis. The physiological consequences of both targeting routes were elucidated in cells lacking the mitochondrial ATP‐binding cassette transporter ATM1, which is essential for biogenesis of cytosolic iron‐sulfur proteins. The mitochondrial MDL1 complex can complement ATM1 function, whereas the endoplasmic reticulum‐targeted version, as well as MDL1 mutants deficient in ATP binding and hydrolysis, cannot overcome the Δatm1 growth phenotype.