To determine the role of the cytoplasmic carboxyl termini of human B1 and B2 kinin receptors (B1KR and B2KR, respectively) in the internalization of their respective ligands, des-Arg10-kallidin and bradykinin (BK), both wild type receptors, as well as truncated B2KRs, a mutated B2KR, and chimeric receptors were stably expressed in Chinese hamster ovary cells. Incubation of [3H]BK at 37 °C with cells expressing wild type B2KR resulted in pronounced and rapid ligand internalization (~80% after 10 min). By contrast, incubation of 3H-labeled des-Arg10-kallidin with cells expressing B1KR resulted in a modest, slow internalization of the ligand (324, of the cytoplasmic carboxyl terminus of the B2KR with that of the B1KR from Cys330 (both Cys residues are putative palmitoylation sites) greatly reduced ligand internalization ~40% after 10 min) without significantly altering K(d) or ligand-induced signal activation. By marked contrast, the corresponding replacement, of the sequence from Cys330 of the cytoplasmic carboxyl terminus of the B1KR with the segment of the B2KR, led to a striking increase of ligand internalization (~75% within 10 min) without altering K(d) or ligand-induced signal activation. Truncation of the B2KR to within three amino acids of Cys324 (truncation at Gly327 led to strongly reduced ligand internalization (~40% after 10 min). Truncation of the B2KR up to Lys315 almost completely abolished internalization of [3H]BK (10% after 10 min). This additional reduction is apparently not caused by the loss of the potential palmitoylation site at Cys324, since a B2KR with a point mutation of Cys324 to Ala internalized [3H]BK as rapidly as the wild type B2KR. From these results we conclude that the cytoplasmic carboxyl terminus of the human B2KR contains sequences that are necessary and sufficient to permit rapid ligand-induced sequestration of human kinin receptors and internalization of their agonists.
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