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J Biol Chem ; 271(3): 1748-55, 1996 01 19.
Article in English | MEDLINE | ID: mdl-8576178

ABSTRACT

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised. The antibodies strongly reacted with their corresponding antigens and cross-reacted both with the denatured and the native B2 receptor. Affinity-purified antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140. Antibodies to extracellular domain 3 (second loop) efficiently interfered, in a concentration-dependent manner, with agonist and antagonist binding and vice versa. Antibodies to extracellular domain 4 (third loop) blocked binding of the agonist but not of the antagonist, whereas antibodies to extracellular domains 1 and 2 or to intracellular domains failed to block ligand binding. Antibodies to ectodomain 3 competed with agonistic anti-idiotypic antibodies for B2 receptor binding. Further, affinity-purified antibodies to the amino-terminal portion of extracellular domain 3 transiently increased intracellular free Ca2+ concentration and thus are agonists. The Ca2+ signal was specifically blocked by the B2 antagonist HOE140. By contrast, antibodies to the carboxyl-terminal segment of extracellular domain 4 failed to trigger Ca2+ release. The specific effects of antibodies to the amino-terminal portion of extracellular domain 3 suggest that this portion of the B2 receptor may be involved in ligand binding and in agonist function.


Subject(s)
Protein Structure, Secondary , Receptors, Bradykinin/chemistry , Receptors, Bradykinin/metabolism , Amino Acid Sequence , Animals , Antibodies/pharmacology , Bradykinin/analogs & derivatives , Bradykinin/metabolism , CHO Cells , Cell Line , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cricetinae , Fluorescent Antibody Technique , Humans , Models, Structural , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/immunology , Rats , Receptor, Bradykinin B2 , Receptors, Bradykinin/agonists , Recombinant Proteins/agonists , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera , Transfection
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