B2 kinin receptor-mediated internalization of bradykinin in DDT1 MF-2 smooth muscle cells is paralleled by sequestration of the occupied receptors.

We have previously shown that exposure of DDT1 MF-2 smooth muscle cells to the agonist bradykinin (BK) results in a rapid B2 kinin receptor-mediated internalization of BK followed by degradation of the intracellular BK [Munoz, C. M., and Leeb-Lundberg, L. M. F. (1992) J. Biol. Chem. 267, 303-309]. Here, we show that BK internalization is paralleled by sequestration of the occupied B2 receptors. Sequestration was observed as a stoichiometric decrease in the number of accessible B2 receptors, i.e., the binding of one molecule of BK effectively compartmented that receptor so as to render the binding site unavailable to the extracellular environment. The rate at which BK stimulated sequestration (t1/2 approximately 7 min) was almost the same as that for BK internalization (t1/2 approximately 9 min). Agonist specificity for the receptor sequestration was indicated by the lack of effect by the high affinity B2 kinin receptor-specific antagonist [D-Arg0, Hyp3, D-Phe7, Thi5,8]-BK (10 microM). Removal of free and bound extracellular BK resulted in a rapid (t1/2 approximately 15 min) reappearance of accessible receptors and this process was not sensitive to the protein synthesis inhibitor cycloheximide. Essentially all of the cellular receptors identified by BK were associated with the plasma membrane fraction. A majority of the sequestered receptors remained inaccessible following cell disruption. However, sequestered receptors were retrievable by treating particulate fractions with the detergent 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid. Pretreatment with BK (1 microM), the alpha 1-adrenergic agonist norepinephrine (10 microM), or the tumor promoting phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) resulted in desensitization of BK stimulation of phosphatidylinositol (PI) turnover. On the other hand, only BK stimulated B2 receptor sequestration. Together, these results suggest that B2 kinin receptors become sequestered by the cell in a compartment contiguous with the plasma membrane following occupancy of the receptor by an agonist. This sequestration appears to be closely associated with BK internalization and not a general mechanism for desensitization of BK-stimulated PI turnover.