title: Alanine screening of the intracellular loops of the human bradykinin B receptor--effects on receptor maintenance, G protein activation and internalization.
abstract: The bradykinin B(2) receptor is coupled to G protein G(q/11) and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY --> AAA, retained the ability to specifically bind [(3)H]bradykinin. The binding affinities at 4 or 37 degrees C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [(3)H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in G(q/11) activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability.
authors.affiliation: Ludwig-Maximilians-Universität München, Abteilung für Klinische Chemie und Klinische Biochemie, Muenchen, Germany. alexander.faussner@med.uni-muenchen.de
authors.forename: Alexander
authors.initials: A
authors.lastname: Faussner
authors.forename: Goeran
authors.initials: G
authors.lastname: Wennerberg
authors.forename: Steffen
authors.initials: S
authors.lastname: Schüssler
authors.forename: Jens
authors.initials: J
authors.lastname: Feierler
authors.forename: Cornelia
authors.initials: C
authors.lastname: Seidl
authors.forename: Marianne
authors.initials: M
authors.lastname: Jochum
authors.forename: David
authors.initials: D
authors.lastname: Proud
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001920:Bradykinin / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011487:Protein Conformation; D043782:Receptor, Bradykinin B2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007295:Inositol Phosphates; D043782:Receptor, Bradykinin B2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000409:Alanine; D001920:Bradykinin