Nuclear localization of bradykinin B2 receptors reflects binding to the nuclear envelope protein lamin C

Abstract The mechanism of action of bradykinin (BK), a pro-inflammatory mediator, is thought to be mediated by specific cell surface membrane bradykinin B 2 receptors. Some evidence suggests that there are both intracellular and nuclear bradykinin B 2 receptors. This study identified proteins that interact with the C-terminus of the bradykinin B 2 receptor (in particular, the nuclear membrane protein lamin C), using the yeast two-hybrid system. The motif of the C-terminal domain (CT) mutant 303–320 in bradykinin B 2 receptor was identified as a lamin C protein binding motif. Immunohistochemistry revealed colocalization of FLAG- bradykinin B 2 receptor with HA-lamin C in the nucleus of HEK 293T cells. In situ proximity ligation assay (PLA) showed that FLAG-bradykinin B 2 receptor formed heterodimers with HA-lamin C in the nucleus. In addition, live cell fluorescence imaging showed that bradykinin B 2 receptor-EGFP was located in the nucleus and co-localized with HcRed-lamin C. Interestingly, neither BK addition nor bradykinin B 2 receptor CT mutation reduced the binding to lamin C or changed the distribution of bradykinin B 2 receptor. Taken together, these findings demonstrate that bradykinin B 2 receptor-lamin C heterodimers form in the nucleus independent of BK stimulation and CT mutation. We propose that heterodimerization of bradykinin B 2 receptor with lamin C is essential to nuclear localization of bradykinin B 2 receptor and plays an important role in cell signaling and function.