Structural features of the human bradykinin B2 receptor probed by agonists, antagonists, and anti-idiotypic antibodies.
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.
Institute for Physiological Chemistry and Pathobiochemistry, University of Mainz, Germany.
S A
SA
Alla
J
J
Buschko
U
U
Quitterer
A
A
Maidhof
M
M
Haasemann
G
G
Breipohl
J
J
Knolle
W
W
Müller-Esterl
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D001920:Bradykinin / Q000378:metabolism*; D002478:Cells, Cultured; D003429:Cross Reactions; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D001920:Bradykinin