title: Kinin B2 receptor-mediated bradykinin internalization and metalloendopeptidase EP24.15-dependent intracellular bradykinin degradation.
abstract: 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 B(2) receptor (B(2)R) 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 B(2)R. B(2)R 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 B(2)R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B(2)R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca(2+) mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B(2)R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B(2)R responsiveness, and it serves as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.
authors.affiliation: Department of Experimental Medical Science, Lund University, SE-22184 Lund, Sweden.
authors.forename: Caroline
authors.initials: C
authors.lastname: Sandén
authors.forename: Johan
authors.initials: J
authors.lastname: Enquist
authors.forename: Sara H
authors.initials: SH
authors.lastname: Bengtson
authors.forename: Heiko
authors.initials: H
authors.lastname: Herwald
authors.forename: L M Fredrik
authors.initials: LM
authors.lastname: Leeb-Lundberg
mesh_terms: D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D001920:Bradykinin / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000502:physiology*
chemical_list: D043782:Receptor, Bradykinin B2; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D001920:Bradykinin