The sixth transmembrane domains of the human B1 and B2 bradykinin receptors are structurally compatible and involved in discriminating between subtype-selective agonists.
In order to investigate the molecular basis for the ability of the human B1 and B2 bradykinin (BK) receptor subtypes to discriminate between subtype-selective ligands, we constructed chimeric proteins in which the sixth transmembrane domains (TM-VI) of these receptors were exchanged. The pharmacological profiles of the constructs were analyzed by radioligand binding in particulate preparations of transiently transfected HEK293 cells using the agonist [3H]des-Arg10-kallidin and the antagonist [3H]NPC17731. The ability of these constructs to transmit an intracellular signal was measured in transiently transfected A10 cells, a vascular smooth muscle cell line, by single cell Ca2+ imaging. Substitution of B1 TM-VI into the B2 receptor (B2(B1VI)) dramatically reduced the affinity of the B2-selective agonist BK, whereas the affinity of the B2-selective antagonist NPC17731 was unaltered. High affinity BK binding was fully regained when two residues, Tyr259 and Ala263, near the extracellular surface of TM-VI in B2(B1VI), were replaced with the corresponding residues in the wild-type B2 receptor, which are Phe259 and Thr263. The construct B1(B2VI), produced by substitution of B2 TM-VI into the B1 receptor, did not support high affinity binding of the B1-selective agonist des-Arg10-kallidin. In contrast to BK and des-Arg10-kallidin, the binding of the less subtype-selective agonist kallidin showed little sensitivity to TM-VI exchange. These results show that TM-VI in the human B1 and B2 BK receptor subtypes, although only 36% identical, are structurally compatible. Furthermore, this domain contributes significantly to the ability of these receptors to discriminate between the subtype-selective agonists BK and des-Arg10-kallidin.
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Department of Biochemistry, The University of Texas Health Science Center, San Antonio 78284-7760, USA.
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D001667:Binding, Competitive; D002460:Cell Line; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D011869:Radioligand Assay; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D008024:Ligands; D008565:Membrane Proteins; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins