INFLUENCE OF THE CYTOSOLIC CARBOXYL TERMINI OF HUMAN B1 AND B2 KININ RECEPTORS ON RECEPTOR SEQUESTRATION, LIGAND INTERNALIZATION, AND SIGNAL-TRANSDUCTION
A. Faussner et al., INFLUENCE OF THE CYTOSOLIC CARBOXYL TERMINI OF HUMAN B1 AND B2 KININ RECEPTORS ON RECEPTOR SEQUESTRATION, LIGAND INTERNALIZATION, AND SIGNAL-TRANSDUCTION, The Journal of biological chemistry, 273(5), 1998, pp. 2617-2623
To determine the role of the cytoplasmic carboxyl termini of human B1
and B2 kinin receptors (B1KR and B2KR, respectively) in the internaliz
ation of their respective ligands, des-Arg(10)-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 [H-3]BK at 37 degrees C with cells express
ing wild type B2KR resulted in pronounced and rapid ligand internaliza
tion (similar to 80% after 10 min), By contrast, incubation of H-3-lab
eled des-Arg(10)-kallidin with cells expressing B1KR resulted in a mod
est, slow internalization of the Ligand (<20% after 10 min), Replaceme
nt, from Cys(324), Of the cytoplasmic carboxyl terminus of the B2KR wi
th that of the B1KR from Cys(330) (both Cys residues are putative palm
itoylation sites) greatly reduced ligand internalization (similar to 4
0% after 10 min) without significantly altering K-d or ligand-induced
signal activation, By marked contrast, the corresponding replacement,
of the sequence from Cys(330) Of the cytoplasmic carboxyl terminus of
the B1KR with the segment of the B2KR, led to a striking increase of l
igand internalization (similar to 75% within 10 min) without altering
K-d Or ligand-induced signal activation. Truncation of the B2KR to wit
hin three amino acids of Cys(324) (truncation at Gly(327)) led to stro
ngly reduced ligand internalization (similar to 40% after 10 min), Tru
ncation of the B2KR up to Lys(315) almost completely abolished interna
lization of [H-3]BK (10% after 10 min). This additional reduction is a
pparently not caused by the loss of the potential palmitoylation site
at Cys(324), since a B2KR with a point mutation of Cys(324) to Ala int
ernalized [H-3]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 l
igand-induced sequestration of human kinin receptors and internalizati
on of their agonists.