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Alanine screening of the intracellular loops of the human bradykinin B₂ receptor - effects on receptor maintenance, G protein activation and internalization

Faussner, Alexander, Wennerberg, Goeran, Schüssler, Steffen, Feierler, Jens, Seidl, Cornelia, Jochum, Marianne, Proud, David
FEBS journal 2009 v.276 no.13 pp. 3491-3503
alanine, antagonists, binding capacity, binding sites, bradykinin, humans, hydrolysis, mutants, point mutation, screening, tritium
The bradykinin B₂ receptor is coupled to G protein Gq/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 [rightward arrow] AAA, retained the ability to specifically bind [³H]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 [³H]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 Gq/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.