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Alanine screening of the intracellular loops of the human bradykinin B 2 receptor – effects on receptor maintenance, G protein activation and internalization
Author(s) -
Faussner Alexander,
Wennerberg Goeran,
Schüssler Steffen,
Feierler Jens,
Seidl Cornelia,
Jochum Marianne,
Proud David
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07071.x
Subject(s) - internalization , allosteric regulation , mutant , receptor , intracellular , bradykinin receptor , alanine , bradykinin , biochemistry , biology , ligand (biochemistry) , wild type , microbiology and biotechnology , chemistry , amino acid , gene
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 °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.