Human bradykinin B2 receptors isolated by receptor-specific monoclonal antibodies are tyrosine phosphorylated.
We report the immunoaffinity isolation of bradykinin B2 receptors in a tyrosine-phosphorylated state from WI-38 human lung fibroblasts. We generated six monoclonal antibodies directed against B2 bradykinin receptor biologic activity mediating prostaglandin E2 production in WI-38. These cells express a repertoire of bradykinin receptor affinity forms with closely correlated biologic activity and [3H]bradykinin binding. Some of the monoclonal antibodies selectively recognize intermediate-affinity (Kd = 5.6 nM) or low-affinity (Kd = 42 nM) receptor forms, whereas others recognize epitopes common to both. The monoclonal antibodies block bradykinin binding and biologic activity. Immunoaffinity chromatography on an immobilized monoclonal antibody of intermediate- plus low-affinity specificity yields WI-38 B2 receptors with intact [3H]bradykinin binding activity and a molecular mass of 78 kDa. The same band is immunoblotted by all the monoclonal antibodies, indicating a similar molecular mass for receptor forms of different affinity. Anti-phosphotyrosine antibodies demonstrate that the receptors are tyrosine phosphorylated, with implications for receptor function and regulation. Genistein completely inhibits bradykinin-mediated prostaglandin E2 production with an IC50 of 8 microM, indicating that tyrosine kinase activity is critical for the signal transduction leading to arachidonic acid release.
Jong
Y J
YJ
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110.
Dalemar
L R
LR
Wilhelm
B
B
Baenziger
N L
NL
D000911:Antibodies, Monoclonal / Q000276:immunology; D001920:Bradykinin / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015232:Dinoprostone / Q000096:biosynthesis; D019833:Genistein; D006801:Humans; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D019000:Phosphotyrosine; D018002:Receptors, Bradykinin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007529:Isoflavones; D018002:Receptors, Bradykinin; D019000:Phosphotyrosine; D014443:Tyrosine; D019833:Genistein; D015232:Dinoprostone; D001920:Bradykinin