Late Endosomal/Lysosomal Targeting and Lack of Recycling of the Ligand-Occupied Endothelin B Receptor
- Alexander Oksche1,
- Gregor Boese1,
- Angelika Horstmeyer1,
- Jens Furkert1,
- Michael Beyermann1,
- Michael Bienert1 and
- Walter Rosenthal1,2
- 1Forschungsinstitut für Molekulare Pharmakologie (A.E., G.B., A.H., J.F., M.Be., M.Bi., W.R.) and 2Institut für Pharmakologie, Freie Universität Berlin (W.R.), Berlin, Germany
Abstract
A fusion protein consisting of the endothelin B (ETB) receptor and the enhanced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ETB receptor. The ETBreceptor and the ETB/EGFP fusion protein displayed very similar pharmacological properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temperature PAGE analysis of the 125I-ET1-bound ETB receptor and the 125I-ET1-bound ETB/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ETB/EGFP fusion protein demonstrated strong signals at the plasma membrane. On addition of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incubation, ETB/EGFP and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using 125I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ETB receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of protease-activated receptors.
Footnotes
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Send reprint requests to: Dr. Alexander Oksche, Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke-Str. 4, D-10315 Berlin. E-mail:oksche{at}fmp-berlin.de
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The work was supported by the Deutsche Forschungsgemeinschaft (FG 341) and the Fonds der Chemischen Industrie.
- Abbreviations:
- ET1
- endothelin 1
- ET2
- endothelin 2
- ET3
- endothelin 3
- ETA
- human endothelin A
- ETB
- human endothelin B
- GPCR
- G protein-coupled receptor
- GRK
- G protein-coupled receptor kinase
- CHO
- Chinese hamster ovary
- EGFP
- enhanced green fluorescent protein
- LDL
- low-density lipoproteins
- LH/hCG receptor
- luteinizing hormone/human choriogonadotropin receptor
- PAR
- protease-activated receptor
- TRITC
- tetramethylrhodamine isothiocyanate
- Tfr
- transferrin
- DiI
- 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
- Cy3
- Cyanin3
- Fluo
- 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester
- LT-PAGE
- low temperature-polyacrylamide gel electrophoresis
- BAME
- bacitracin/aprotinin/MgCl2/EGTA
- DPBS
- Dulbecco's PBS
- NK1
- neurokinin 1
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- Received December 29, 1999.
- Accepted February 25, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
