Regulation and Intracellular Trafficking Pathways of the Endothelin Receptors

• Published in: The Journal of biological chemistry 2000-06, Vol.275 (23), p.17596-17604
• Main Authors: Bremnes, Toril, Paasche, Joachim D., Mehlum, Anja, Sandberg, Cecilie, Bremnes, Bjørn, Attramadal, Håvard
• Format: Article
• Language: English
• Subjects: Animals; Arrestins - physiology; beta-Adrenergic Receptor Kinases; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins; Cell Nucleus - physiology; CHO Cells; COS Cells; Cricetinae; Cyclic AMP-Dependent Protein Kinases - physiology; Dynamins; Endothelin-1 - pharmacology; Endothelin-1 - physiology; Green Fluorescent Proteins; GTP Phosphohydrolases - physiology; Humans; Kinetics; Luminescent Proteins - analysis; Luminescent Proteins - genetics; Lysosomes - physiology; Mutagenesis, Site-Directed; Phosphatidylinositols - metabolism; Phosphorylation; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin - physiology; Recombinant Fusion Proteins - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000142200

The effects of endothelin (ET) are mediated via the G protein-coupled receptors ETA and ETB. However, the mechanisms of ET receptor desensitization, internalization, and intracellular trafficking are poorly understood. The aim of the present study was to investigate the molecular mechanisms of ET receptor regulation and to characterize the intracellular pathways of ET-stimulated ETA and ETB receptors. By analysis of ETA and ETB receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed βARK, β-arrestin-1, β-arrestin-2, or dynamin as well as dominant negative mutants of these regulators, we have demonstrated that both ET receptor subtypes follow an arrestin- and dynamin/clathrin-dependent mechanism of internalization. Fluorescence microscopy of Chinese hamster ovary and COS cells expressing green fluorescent protein (GFP)-tagged ET receptors revealed that the ETA and ETB subtypes were targeted to different intracellular routes after ET stimulation. While ETA-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ETB-GFP was targeted to lysosomes after ET-induced internalization. Both receptor subtypes colocalized with Rab5 in classical early endosomes, indicating that this compartment is a common early intermediate for the two ET receptors during intracellular transport. The distinct intracellular routes of ET-stimulated ETA and ETB receptors may explain the persistent signal response through the ETAreceptor and the transient response through the ETBreceptor. Furthermore, lysosomal targeting of the ETBreceptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.