Abstract

We investigated the subcellular localization of two endothelin receptors (ET(A)R and ET(B)R). To visualize these receptors directly, the C terminus of each receptor was fused to the N terminus of enhanced green fluorescent protein (designated as ETR-EGFP). When transiently expressed in various mammalian cell lines, ET(A)R-EGFP was predominantly localized on the plasma membrane. By contrast, ET(B)R-EGFP was, independent of ligand stimulation, predominantly localized on the intracellular vesicular structures containing Lamp-1. Immunoblot analyses revealed that at steady state ET(B)R-EGFP was highly degraded, and its degradation was inhibited by bafilomycin A(1). Antibody uptake experiments suggested that the ET(B)R-EGFP molecules were internalized from the plasma membrane. It is therefore likely that ET(B)R is first transported to the plasma membrane and then internalized, irrespective of ligand stimulation, to lysosomes where it undergoes proteolytic degradation. Exchanging the C-terminal cytoplasmic tails of the two ETRs revealed that the cytoplasmic tail is responsible for both the intracellular localization and the degradation of the receptors. Deletion of the extreme C-terminal 35 amino acids from both receptors allowed the receptor proteins to localize predominantly in the intracellular vesicles and to degrade. These observations indicate that the cytoplasmic tail of ET(A)R determines its plasma membrane localization. Stimulation with endothelin-1 increased the amount of intact ETR-EGFP fusion proteins without increasing their de novo synthesis, suggesting that binding of endothelin-1 stabilizes the ETRs.

Highlights

  • Endothelins are a family of 21-amino acid peptides consisting of three isopeptides as follows: endothelin-1, endothelin-2, and endothelin-3 (ET-1,1 ET-2, and ET-3, respectively) (1, 2)

  • The most characterized GPCR is ␤2-adrenergic receptor. When it is stimulated with its agonist, the serine and threonine residues in the cytoplasmic tail are phosphorylated by G protein-coupled receptor kinases and cAMP-dependent kinase, followed by its association with ␤-arrestins, resulting in uncoupling to G proteins or desensitization. ␤-Arrestins play a crucial role in the ligand-dependent internalization of this receptor, mediated by clathrin-coated vesicles

  • Difference in the Subcellular Localization between endothelin-A receptor (ETAR) and endothelin-B receptor (ETBR)—To visualize the intracellular localization of two subtypes of the endothelin receptor, ETAR and ETBR, the C terminus of each receptor was fused to the N terminus of enhanced green fluorescent protein (EGFP)

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Summary

EXPERIMENTAL PROCEDURES

Rat clone 9 cells were maintained in DMEM/F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Fortyeight hours after transfection, cells were fixed with cold methanol, permeabilized with 0.1% Triton X-100 in PBS, and incubated with rabbit polyclonal anti-rat Lamp-1 antibody (kindly provided by Dr Kenji Akasaki, Fukuyama University, Japan) for clone 9 or mouse monoclonal anti-human Lamp-1 antibody (PharMingen) for HeLa cells for 1 h at room temperature, followed by labeling with Cy3-conjugated goat antibodies against rabbit or mouse IgG, respectively (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. For analysis of endocytosis of ETBR, clone 9 cells or HeLa cells transfected with the ETBR-EGFP plasmid were incubated with 5 ␮g/ml rabbit anti-peptide antibody against the N-terminal region of human ETBR (amino acids 27– 42) (IBL) for 24 h simultaneously with transfection. Values were represented as percent of maximal response of noradrenaline (10 ␮M) stimulation

RESULTS
Cellular Trafficking of Endothelin Receptors
DISCUSSION
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