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CELLULAR AND MOLECULAR

Kinin B₂ Receptor-Mediated Bradykinin Internalization and Metalloendopeptidase EP24.15-Dependent Intracellular Bradykinin Degradation

Unit of Drug Target Discovery, Division of Cellular and Molecular Pharmacology, Department of Experimental Medical Science (C.S., J.E., L.M.F.L.-L.), and Division of Infectious Medicine, Department of Clinical Science (S.H.B., H.H.), Lund University, Lund, Sweden

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 3.4.24.1 [EC] 5; 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.

Address correspondence to: Dr. L. M. Fredrik Leeb-Lundberg, Department of Experimental Medical Science, Lund University, BMC, A12, SE-22184 Lund, Sweden. E-mail: fredrik.leeb-lundberg{at}med.lu.se