The effects of endothelin (ET) are mediated via the G protein-coupled receptors ET(A) and ET(B). 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 ET(A) and ET(B) receptors. By analysis of ET(A) and ET(B) receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed betaARK, 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 ET(A) and ET(B) subtypes were targeted to different intracellular routes after ET stimulation. While ET(A)-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ET(B)-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 ET(A) and ET(B) receptors may explain the persistent signal response through the ET(A) receptor and the transient response through the ET(B) receptor. Furthermore, lysosomal targeting of the ET(B) receptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.
We recently reported that the endothelin (ET) receptor subtypes ET 275, 17596 -17604). The ET A receptor was shown to follow the recycling route of transferrin, whereas ET B is targeted to lysosomes for degradation. In the present study we have investigated the mechanisms of ET receptor subtype-specific targeting to distinct intracellular trafficking pathways. Truncation mutants of the ET A and ET B receptors with deletions of the cytoplasmic carboxyl-terminal tail distal to the palmitoylation site were found to mediate inositol phosphate accumulation and to internalize upon agonist stimulation, although internalization occurred at a slower rate as compared with the wild-type receptors. However, the truncated ET A receptor was no longer able to undergo recycling. Rather, both truncation mutants were recognized by -arrestin for recruitment to endocytosis and were sorted to lysosomes by a dynamin-dependent internalization pathway. Furthermore, studies of chimeric ET A and ET B receptors where the cytoplasmic tail of ET A was swapped with the corresponding domain of ET B , and vice versa, revealed that the cytoplasmic tail of ET B is required for efficient lysosomal sorting and that signals for targeting to recycling reside in the cytoplasmic tail of the ET A receptor.The multiple physiological effects of the vasoactive peptide endothelin (ET) 1 (1) are mediated by the G protein-coupled receptors (GPCRs) ET A and ET B (2). In the vasculature, ET A receptors residing on the smooth muscle cells mediate prolonged vasoconstriction (3), whereas ET B receptors, which are on the plasma membrane of endothelial cells, are primarily considered to cause NO-mediated vasodilatation (4). In addition, considerable evidence now also supports a role for ET B receptors in the clearance of plasma ET-1 from the circulation (5-8). In order to elucidate the molecular mechanisms of these distinct physiological responses, we recently characterized the intracellular trafficking pathways of the ET A and ET B receptors (9). Upon agonist stimulation both receptor subtypes are rapidly internalized by mechanisms that depend on G proteincoupled receptor kinase, arrestin, clathrin, and dynamin. Interestingly, the internalized ET A and ET B receptors initially appear to share a common path into Rab5-positive early endosomes. However, the two receptor subtypes are subsequently targeted to different intracellular fates. Whereas the ET A receptor follows the recycling pathway through the pericentriolar recycling compartment and reappears at the plasma membrane, the ET B receptor is directed to lysosomes for degradation. In terms of physiological effects, rapid recycling of the ET A receptor may provide the basis for reestablishment of the signaling response, and thus for the sustained vasoconstriction mediated through this receptor. Conversely, lysosomal targeting of the ET B receptor is consistent with a role for this receptor subtype in the clearance of ET-1 from the circulation. In this respect, it was recently also demonstrated that ET-1 is cotr...
We have previously reported that endocytic sorting of ET A endothelin receptors to the recycling pathway is dependent on a signal residing in the cytoplasmic carboxyl-terminal region. The aim of the present work was to characterize the carboxylterminal recycling motif of the ET A receptor. Assay of truncation mutants of the ET A receptor with increasing deletions of the carboxyl-terminal tail revealed that amino acids 390 to 406 contained information critical for the ability of the receptor to recycle. This peptide sequence displayed significant sequence similarity to several protein segments confirmed by X-ray crystallography to adopt antiparallel -strand structures (-finger). One of these segments was the -finger motif of neuronal nitric-oxide synthase reported to function as an internal PDZ (postsynaptic density-95/disc-large/zona occludens) domainbinding ligand. Based on these findings, the three-dimensional structure of the recycling motif of ET A receptor was predicted to attain a -finger conformation acting as an internal PDZ ligand. Site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative -finger conformation or PDZ ligand function prevented recycling of the ET A receptor. Analysis of more than 300 G protein-coupled receptors (GPCRs) identified 35 different human GPCRs with carboxylterminal sequence patterns that fulfilled the structural criteria of an internal PDZ ligand. Among these are several receptors reported to follow a recycling pathway. In conclusion, recycling of ET A receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This structural motif may represent a more general principle of endocytic sorting of GPCRs.The physiological effects of the vasoactive peptide endothelin-1 (ET-1) are mediated by the ET A and ET B receptors, which belong to class A G protein-coupled receptors (GPCRs) (Yanagisawa et al., 1988). In the vasculature, ET A receptors residing on smooth muscle cells mediate prolonged vasoconstriction, whereas ET B receptors, which are located on the plasma membrane of endothelial cells, are primarily considered to cause NO-mediated vasodilation (Yanagisawa and Masaki, 1989). In addition, considerable evidence now also supports a role for the ET B receptor in clearance of plasma ET-1 from the circulation (Berthiaume et al., 2000;Opgenorth et al., 2000). We have previously shown that agonist-induced internalization of the two receptor subtypes depends on a mechanism involving G protein-coupled receptor kinase, arrestin, clathrin, and dynamin (Bremnes et al., 2000). After internalization, however, the two receptor subtypes are targeted to different intracellular fates. The ET A receptor follows the recycling pathway through the pericentriolar recycling compartment and subsequently reappears on the plasma membrane, whereas the ET B receptor is directed to lysosomes for degradation (Bremnes et al., 2000). In terms of physiological effects, rapid recycling of the ET A receptor may provide ba...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.