Helix 8 plays a crucial role in bradykinin B(2) receptor trafficking and signaling.
Upon activation the human bradykinin B(2) receptor (B(2)R) acts as guanine nucleotide exchange factor for the G proteins G(q/11) and G(i). Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits β-arrestins, which block further G protein activation and promote B(2)R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B(2)R. We show here that disruption of helix 8 in the B(2)R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B(2)R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B(2)R with GRK2/3 and β-arrestin2 as shown by co-immunoprecipitation. Recruitment of β-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B(2)R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B(2)R with GRK2/3 and β-arrestins. Additional data obtained with chimera of B(2)R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.
Abteilung für Klinische Chemie und Klinische Biochemie, Ludwig-Maximilians-Universität, Nussbaumstrasse 20, D-80336 München, Germany.
Jens
J
Feierler
Markus
M
Wirth
Benjamin
B
Welte
Steffen
S
Schüssler
Marianne
M
Jochum
Alexander
A
Faussner
D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000235:genetics / Q000502:physiology*; D043782:Receptor, Bradykinin B2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship
D007295:Inositol Phosphates; D043782:Receptor, Bradykinin B2