Intramolecular signal transduction by the bradykinin B2 receptor.
To study the intramolecular signal transduction, we performed single point and cassette mutations in transmembranal and intracellular regions of the bradykinin B2 receptor. We studied the influence of the two intramembranal Cys residues at positions 304 and 348, the role of Arg at position 177 in the highly conserved tripeptide sequence Asp-Arg-Tyr, the cytosolic G-protein binding area, and attempted to verify the general hypothesis of an ion tunnel-like interface in GPCRs. Wild type receptor, His-tagged receptor, and His-tagged mutant receptors were expressed in COS-7 cells and functionally compared by bradykinin-induced formation of inositolphosphate and arachidonic acid. To investigate the expression, all mutants were modified at the N-terminus by insertion of two successive His-tags and detected with an anti-poly-His antibody. Replacement of the second and third cytosolic loop by a loop from another membrane protein as well as single replacement of Arg at position 177 by Ala leads to a fully inactive receptor mutant without any ligand binding affinity and stimulatory activity. Mutants with replacement of Cys residues 304 and 348 by Ser showed only moderate effects. Regardless of the replacement of Asp 407 by Ala, the receptor is able to increase the agonist-induced levels of inositolphosphate and of arachidonic acid, indicating that our studies can not verify the postulated ion tunnel hypothesis.
Mueller
Sylvia
S
Institute of Biochemistry and Biophysics, Friedrich-Schiller-University, Philosophenweg 12, 07743 Jena, Germany.
Liebmann
Claus
C
Reissmann
Siegmund
S
D019943:Amino Acid Substitution; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011869:Radioligand Assay; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D016718:Arachidonic Acid