Abstract
The human bradykinin B2 receptor belongs to the family of G-protein-coupled receptors. To characterize the receptor protein, we have solubilized the membranes of cultured human foreskin fibroblasts bearing the B2 receptor. Affinity cross-linking of the solubilized receptor with the labeled agonist, 125I-Tyr0-bradykinin, or the labeled antagonist, 125I-(4-hydroxy-phenyl-propionyl)-HOE140, revealed major bands of apparent molecular mass of 69 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and of 59 kDa under non-reducing conditions. A 1000-fold molar excess of each of the unlabeled ligands quenched the specific labeling suggesting that the agonist and the antagonist compete for overlapping binding site(s). Covalent coupling of the receptor to bradykinin or HOE140, followed by Western blotting and immunoprinting with specific anti-ligand antibodies confirmed that the major ligand-binding form of the receptor is of 69 kDa. Anti-idiotypic antibodies which bear the internal image of bradykinin (Haasemann, M., Buschko, J., Faussner, A., Roscher, A.A., Hoebeke, J., Burch, R.M., and Müller-Esterl, W. (1991) J. Immunol. 147, 3882-3892) immunoprecipitated the 125I-labeled receptor as a major band of 68 kDa and a minor band of 47 kDa indicative of partial proteolysis. Chemical deglycosylation of the 125I-labeled receptor shifted the apparent molecular mass from 69 to 44 kDa demonstrating that the receptor is heavily glycosylated. Two-dimensional electrophoresis of the affinity-purified receptor revealed overlapping spots of 69 kDa and of pI 6.8-7.1 pointing to a microheterogeneity of the carbohydrate moiety. Elucidation of the key structural features of the B2 receptor protein will aid in understanding the structure-function relationships governing this prototypic peptide receptor.
Highlights
The human bradykinin B2 receptor belongs to the Silva et al, 1949), mediates importantphysiological family of G-protein-coupled receptors
We have set out tocharacterize the struc- Approximately 30% of the initial receptor binding activity present on tural features of the human B2 receptor protein by affinity the membranes was recovered in the solubilizate, whereas < 5% of cross-linking, immunoprinting, and endogenous labeling the binding activity remained associated with the membrane pellet
Affinity Cross-linking of B2 Receptor-Human fibroblast membranes were solubilized with 4 mM CHAPS, andthe following the original description (Haasemann et al, 1991).The vast resultant preparation incubated with 20 I ~ Mof i25J-Tyro-bra
Summary
(Hock et al, 1990).Monoclonal antibodies were purified from ascites Ligund Assay-The relative affinities of various ligands by 45% (w/v) ammonium sulfateprecipitation followed by DE52cellulose chromatography. The first antibody have an appreciable affinity ( K ,2 100p ~ ) L.ikewise a control was from supernatant of hybridoma cell culture (undiluted) or from mouse ascites (diluted 1:lOO in buffer B containing 0.1% (w/v) BSA and 0.05% (w/v) Tween 20). Labeled goat anti-mouse (anti-rabbit) immunoglobulin (lo6cpm/ml) peptide, angiotensin 11, was without effect ( K ; 2 100 p ~ ) This rank order of Kivalues classifies the kinin receptor from human foreskin fibroblasts as a B2 type in accordance with the previous characterization (Roscher et al, 1983); no evidence was found for the presence of a B1receptor on this cell in 100 rnl of buffer B including 0.1% (w/v) BSA and 0.05% (w/v) type. Affinity Cross-linking of B2 Receptor-Human fibroblast membranes were solubilized with 4 mM CHAPS, andthe following the original description (Haasemann et al, 1991).The vast resultant preparation incubated with 20 I ~ Mof i25J-Tyro-bra-
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