Subtype-specific trafficking of endothelin receptors

We investigated the subcellular localization of two endothelin receptors (E TAR and ETBR), To visualize these receptors directly, the C terminus of eac h receptor was fused to the N terminus of enhanced green fluorescent protei n (designated as ETR-EGFP). When transiently expressed in various mammalian cell lines, ETAR-EGFP was predominantly localized on the plasma membrane. By contrast, ETBR-EGFP was, independent of Ligand stimulation, predominantl y localized on the intracellular vesicular structures containing Lamp-1. im munoblot analyses revealed that at steady state ETBR-EGFP was highly degrad ed, and its degradation was inhibited by bafilomycin A,. Antibody uptake ex periments suggested that the ETBR-EGFP molecules were internalized from the plasma membrane. It is therefore likely that ETBR is first transported to the plasma membrane and then internalized, irrespective of ligand stimulati on, 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 degrad ation of the receptors. Deletion of the extreme C-terminal 35 amino acids f rom both receptors allowed the receptor proteins to localize predominantly in the intracellular vesicles and to degrade. These observations indicate t hat the cytoplasmic tail of ETAR determines its plasma membrane localizatio n. Stimulation with endothelin-l increased the amount of intact ETR-EGFP fu sion proteins without increasing their de novo synthesis, suggesting that b inding of endothelin-l stabilizes the ETRs.