Regulation and intracellular trafficking pathways of the endothelin receptors

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 beta ARK, beta-arrestin-1, beta-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 ETB-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 ETA receptor and the transient response through the ETB receptor. Furthermore, lysosomal targeting of the ETB receptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.