Guanosine nucleotides regulate B2 kinin receptor affinity of agonists but not of antagonists: discussion of a model proposing receptor precoupling to G protein.
The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [3H]BK and the antagonist [3H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0 degrees C, particulate fractions exhibited a single class of binding sites with a Kd of 2.3 nM for [3H]BK and a Kd of 3.8 nM for the antagonist [3H]NPC17731. Incubation with radioligands at 37 degrees C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0-40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37 degrees C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [3H]NPC17731 binding unaffected, but reduced the receptor affinity for [3H]BK to a Kd of about 15 nM. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg2+. It was reversed by incubation at 0 degrees C. The rank order of the guanosine nucleotides for [3H]BK binding reduction was GTP[gammaS] = Gpp[NH]p > GTP = GDP > GDP[betaS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.
Faussner
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A
Dr. von Haunersches Kinderspital der Universität München, Abteilung für Klinische Biochemie, Germany.
Roscher
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AA
D001667:Binding, Competitive / Q000187:drug effects; D001920:Bradykinin; D065168:Bradykinin Receptor Antagonists; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D009711:Nucleotides / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014316:Tritium
D065168:Bradykinin Receptor Antagonists; D006150:Guanine Nucleotides; D009711:Nucleotides; D009842:Oligopeptides; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D014316:Tritium; D006153:Guanosine Diphosphate; C071327:NPC 17731; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D008274:Magnesium; D001920:Bradykinin