TY - JOUR T1 - Kinin B<sub>2</sub> Receptor-Mediated Bradykinin Internalization and Metalloendopeptidase EP24.15-Dependent Intracellular Bradykinin Degradation JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 24 LP - 32 DO - 10.1124/jpet.108.136911 VL - 326 IS - 1 AU - Caroline Sandén AU - Johan Enquist AU - Sara H. Bengtson AU - Heiko Herwald AU - L. M. Fredrik Leeb-Lundberg Y1 - 2008/07/01 UR - http://jpet.aspetjournals.org/content/326/1/24.abstract N2 - Kinins are potent proinflammatory peptides that are produced extracellularly and are rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. In this study, we developed model cell systems expressing the kinin B2 receptor (B2R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) after BK internalization via B2R. B2R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no colocalization with either the endoplasmic reticulum marker calnexin or the Golgi marker GM130. No direct colocalization of B2R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B2R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca2+ mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B2R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B2R responsiveness, and it serves as an intracellular peptidase in the degradation of BK specifically internalized via this receptor. The American Society for Pharmacology and Experimental Therapeutics ER -