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 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.
Footnotes
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This work was supported in part by funds from the Swedish Research Council Grant 15057, AlfredÖsterlunds Stiftelse, and the Vascular Wall Program, Faculty of Medicine at Lund University.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.136911.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; B2R, B2 receptor; BK, bradykinin; ACE, angiotensin I-converting enzyme; B1R, B1 receptor; DMEM, Dulbecco's modified Eagle's medium; FLAG-B2, FLAG-tagged B2R; HA, hemagglutinin; HA-B2, HA-tagged B2R; FLAG-EP, FLAG-tagged EP24.15; HA-EP, HA-tagged EP24.15; IP, immunoprecipitation; coIP, coimmunoprecipitated; IB, immunoblotting; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HDF, high-density fraction; LDMF, low-density microsomal fraction; HPLC, high-performance liquid chromatography; ER, endoplasmic reticulum; PI, phosphoinositide; EC50, agonist concentration that elicits 50% of the maximal response; Emax, maximal agonist response; pEC50, -log10EC50 value.
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↵1 Current affiliation: Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, California.
- Received January 21, 2008.
- Accepted April 21, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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