The role of helix 8 and of the cytosolic C-termini in the internalization and signal transduction of B1 and B2 bradykinin receptors

Abstract

Determinants for desensitization and sequestration of G protein-coupled receptors often contain serine or threonine residues located in their C-termini. The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown. Mutagenesis studies with the B2 bradykinin receptor (B2wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including 1347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist-inducible internalization and the prevention of receptor hypersensitivity. Chimeras of the noninternalizing B1 bradykinin receptor (B1wt) containing these B 2wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required. In fact, further substitution of the B1wt C-terminus with corresponding B2wt regions either at C330(7.71) following putative helix 8 (B1CB2) or at the preceding Y312(7.53) in the NPXXY sequence (B1YB2) resulted in chimeras displaying rapid internalization. Intriguingly, however, exchange performed at K322(7.63) within putative helix 8 generated a slowly internalizing chimera (B1KB2). Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B 1wt to serine (as in B2wt) as being responsible for this effect. The slowly internalizing chimera as well as a B1wt point-mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B1wt or the other chimeras. The slow internalization of B1KB2 was also accompanied by a lack of agonist-induced phosphorylation, that in contrast was observed for B 1YB2 and B1CB2, suggesting that putative helix 8 is either directly or indirectly (e.g. via G protein activation) involved in the interaction between the receptor and receptor kinases.